Published April 8, 2016
Research
SNP-Based MAS in Cotton under DepressedRecombination for Renlon –Flanking Recombinants: Results and Inferences on Wide-Cross Breeding Strategies Xiuting Zheng, Kevin A. Hoegenauer, Jose Quintana, Alois A. Bell, Amanda M. Hulse-Kemp, Robert L. Nichols, and David M. Stelly*
ABSTRACT Strong resistance to reniform nematode (Rotylenchulus reniformis) was previously introgressed from the F-genome diploid species Gossypium longicalyx (2n = 2x = 26) into Upland cotton (G. hirsutum L., 2n = 4x = 52, 2[AD]1), and attributed to the gene Renlon. Two resistant elite lines were released, but their seedlings are differentially stunted in nematode-infested fields. Here, we report on the development of linked SNPs and their use to disrupt “linkage drag” around Renlon. Using advanced backcross-inbred lines with previously identified proximal (PCO) or distal crossover (DCO) events near Renlon, we chose 18 Renlon –linked SNP markers for mapping across two large (880) BC1F1 seed populations. Few recombinants occurred (7 of 1760). Using two of the closest Renlon –linked SNPs to select from a BC1F1 seed population (17,600), we identified 5 homeologous recombinants, but none separated the stunting and resistant phenotypes. We then compared homologous recombination rates using equally sized DCO × PCO INTERCROSS and BACKCROSS populations (n = 88). Many more recombinants occurred among progeny of the INTERCROSS (22) than the BACKCROSS (1). This finding suggests that the best SNP-based strategy for interspecific breeding with the F genome and other homeologous genomes may be to use large-scale MAS to select nearby flanking homeologous recombinants in early generations, map them to identify the two closest PCO and DCO types, then intermate them and use MAS to identify homologous recombinants.
X. Zheng, K.A. Hoegenauer, A.M. Hulse-Kemp, R.L. Nichols, and D.M. Stelly, Dep. of Soil and Crop Sciences, Texas A&M Univ., College Station, TX 77843-2474; J. Quintana and A.A. Bell, USDA-ARS, Southern Plains Agricultural Research Center, Cotton Pathology Research Unit, 2765 F and B Road, College Station, TX 77845; R.L. Nichols, Cotton Incorporated, 6399 Weston Parkway, Cary, NC 27513. Received 19 July 2015. Accepted 10 Dec. 2015. *Corresponding author (
[email protected]). Abbreviations: DCO, distal crossover; FM966, G. hirsutum ‘FiberMax 966’; Gh, G. hirsutum; Gl, G. longicalyx; HLA, (G. hirsutum ´ G. longicalyx) chromosome-doubled ´ G. armourianum; KASP, Kompetitive Allele Specific PCR; LG, linkage group; MAS, marker-assisted selection; PCO, proximal crossover; PCR, polymerase chain reaction; SSR, simple sequence repeat; SNP, single nucleotide polymorphism.
M
odern genetic improvement has increased crop productivity worldwide, but it also has eroded genetic variability of crops (Allard, 1996; Hoisington et al., 1999). Narrowed genetic bases make modern crops vulnerable to environmental stress, diseases, and insects (Chan, 2010; Tanksley and McCouch, 1997). The trends apply as well to cotton (Bowman et al., 1996; Brown, 1983; Brubaker et al., 1999; May et al., 1995; Wendel et al., 1992). More than 95% of cotton fiber is produced from Upland cotton (Gossypium hirsutum L.), the world’s leading natural textile fiber crop. Cotton productivity on many hectares is compromised by the reniform nematode (Rotylenchulus reniformis Linford and Oliveira), an obligate plant parasite that feeds on roots and is a major pest of cotton in the United States and certain other countries (Robinson et al., 2007). Estimated annual losses are ~$130 million in the United States, with major impact in the states of Mississippi, Louisiana, and Alabama (Blasingame, 2006; Koenning et al., 2004; Robinson et al., 2007). Control of reniform nematode has been largely limited to crop rotation and application of nematicides (Stetina et al., 2007). The worsening
Published in Crop Sci. 56:1–14 (2016). doi: 10.2135/cropsci2015.07.0436 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. crop science, vol. 56, july– august 2016
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problem of reniform nematode in cotton production led to a research effort to incorporate genetic resistance to the reniform nematode in Upland cotton genotypes (Nichols et al., 2014). All Upland cotton cultivars lack significant resistance to reniform nematode (Koenning et al., 2004; Robinson et al., 1999; Usery et al., 2005; Weaver et al., 2007), therefore scientists at several laboratories pursued introgression of reniform nematode resistance from other Gossypium species (Avila et al., 2006; Bell et al., 2014, 2015; Robinson et al., 2007; Romano et al., 2009). The African wild species G. longicalyx (Hutch. and Lee, F1 genome, 2n = 26) is the most resistant Gossypium species (Yik and Birchfield, 1984). In histologically characterizing its resistance, Agudelo et al. (2005) observed cell collapse and necrosis in areas where syncytium formation would normally be expected. They suggested that reniform females feeding on G. longicalyx induce a hypersensitive reaction that precludes development of successful feeding sites. Introgression of reniform nematode resistance from G. longicalyx into Upland cotton was achieved by synthesis of a di-species hexaploid, and then a tri-species tetraploid hybrid, “HLA”, [(G. hirsutum × G. longicalyx)2 × G. armourianum Kearn.] (Bell and Robinson, 2004). Strong resistance was introgressed into cotton by backcrossing of the HLA hybrid with G. hirsutum cultivars while exercising phenotypic and/or marker assisted selection (Robinson et al., 2007). Segregation of the reniform nematode resistance suggested monogenic control, and linkage with G. hirsutum chromosome-11 (c11) molecular and phenotypic markers indicated a single dominant gene or haplotype, Renlon (Dighe et al., 2009). Two reniform resistant germplasm lines LONREN-1 and LONREN-2 were subsequently released (Bell et al., 2014). Evaluations of the LONREN lines and derived materials in diverse field environments revealed that plants with the alien Renlon -bearing segment exhibited early season stunting that was pronounced in certain cases when grown in heavily nematode-infested fields, even though they suppressed nematodes. Also, the LONREN and derived resistant lines were shorter and less productive on average than isogenic susceptible lines in spite of their resistance to the nematodes (Bell et al., 2009). Based on examination of roots of severely stunted LONREN germplasm after replicated 60-d treatments of initially sterile soil with ultra-high nematode populations, Sikkens et al. (2011) attributed the differential stunting to hypersensitivity of LONREN to the nematode. However, LONREN lines also exhibit increased genetic susceptibility to certain soil-borne microbial pathogens (Bell et al., 2012). Such susceptibility could also arise by genetic predisposition of LONREN lines and/or due to secondary effects of nematode-related damage at feeding or response sites on the roots (Zheng et al., 2013). Hypothetical explanations for differential stunting of LONREN-1, -2 and other 2
Fig. 1. Primary hypotheses for stunting of LONREN lines in the field. [1] Allelic resistance gene lost. [2] Linkage drag including loss of Gh resistance gene(s) and gain of Gl gene(s) that increase sensitivity to one or more soil pathogens. [3] Pleiotropy effect. [4] Secondary effects, including root damage per se (hypersensitivity to nematodes) and/or increased susceptibility to pathogens.
Renlon lines bred to date are summarized in (Fig. 1). Of special interest here are the hypotheses that Renlon associated linkage drag genetically affects resistance to other soil-borne pathogens, since breakage of such linkages could render Renlon more useful. Many genes important to resistances are known to be clustered and so it would be quite possible that the introgressed Renlon gene for reniform resistance is flanked by other G. longicalyx genes that increase susceptibility to one or more soilborne pathogens. Increased susceptibility and stunting could also result from displacement of one or more G. hirsutum genes that normally would provide resistance to soil-borne pathogen(s), where such genes could be allelic (renhir) or linked to renhir. Given that recombination rates are generally depressed for G. longicalyx chromatin, relative to G. hirsutum germplasm (Dighe et al., 2009), linkage drag would be expected to affect larger chromosomal segments and accordingly larger numbers of syntenic R-genes than if the chromosomes of the source were fully homologous. Regardless of which hypothesis is correct, a high density of Renlon –linked markers is needed to better characterize the Renlon region and various introgression products. In this report, we describe development of a large complement of Renlon –linked markers to better characterize the Renlon-flanking regions and investigate the possible role of Renlon –linked factors as a possible cause for Renlon-associated susceptibility to stunting. We report the discovery, testing and validation of numerous Renlon –linked SNPs between G. longicalyx and G. hirsutum (Gh–Gl SNPs), which we then used for high resolution mapping of the Renlon region. The closest markers were then used for marker-assisted selection of
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Table 1. Pedigree of different LONREN lines.
Plant ID
Resistance source†
Recombinant type‡
Parents/Generation BC2
BC3
BC4
7109
HLA-A132
DCO
Aub 623
DPL 458
DPL 458
STV474
BC5
FM 958
BC6
NemX
BC7
7110
HLA-A122
DCO
NemX
DPL 458
DPL 458
STV454
STV 454
NemX
7111
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV454
FM 958
NemX
7112
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV454
FM 958
NemX
7113
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV454
FM 958
NemX
7114
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV454
FM 958
NemX
7115
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV454
FM 958
NemX
7116
HLA-A84
DCO
DPL 458
DPL 458
DPL 458
STV474
FM 958
NemX
7117
HLA-B45
PCO
DPL 458
DPL 458
FM 958
DPL5415
STV 474
NemX
7118
HLA-A85
PCO
NemX
DPL 458
DPL 458
STV474
FM 958
DPL5415
7119
HLA-B91
PCO
NemX
DPL 458
FM 958
DPL5415
STV 474
NemX
7121
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
STV 474
FM 958
NemX
7122
HLA-A85
DCO
CBL413
DPL 458
STV 454
FM 958
STV 474
NemX
7123
HLA-A85
DCO
NemX
DPL 458
STV 454
FM 958
STV 474
NemX
7124
HLA-A85
DCO
NemX
DPL 458
DPL 458
STV 474
DPL5415
NemX
7125
HLA-B26
DCO
DPL 458
DPL 458
FM 958
DPL 5415
STV474
NemX
7126
HLA-A84
DCO
NemX
DPL 458
FM 966
DPL 5415
DPL5415
DPL493
7127
HLA-A103
DCO
DPL 458
DPL 458
DPL 458
FM 966
FM 966
FM 966
21–2
HLA-A84
With whole Renlon alien segment
Nemx
DPL458
DPL458
DPL458
STV474
FM958
† HLA-B group BC1, pollen from Acala NemX was used to pollinate HLA; HLA-A group BC1, a pool of G. hirsutum pollen from SG125, DP373, PM1220 and TAMCOT Sphinx was used to pollinate HLA. ‡ DCO, distal crossover; PCO, proximal crossover.
recombination events near Renlon. We examined two strategies for securing double recombination events that closely flank Renlon, one based exclusively on homeologous recombination events and the other on a combination of homologous and homeologous recombination events. Lastly, we generalize the discussion about these approaches to marker-assisted selection in the context of wide-cross breeding. In many cases, widecross breeders must selectively introgress potentially valuable genes from genomes that are both replete with agronomically undesirable genes and meiotically distant from the cultivated species’ genome. Two recombination events that closely flank each desired gene/segment are typically required, but depressed rates of recombination can lead to problematically low crossover rates in the region of interest. Fortunately, such situations are becoming more readily addressable as markerassisted approaches to genetic dissection and selection become routine features of breeding.
MATERIALS AND METHODS Germplasm Resources Plant materials included the HLA tri-species hybrid (2n = 4x = 52), the genome of which consists of G. hirsutum ([AD]1), G. longicalyx (F1) as the Renlon donor parent, and a wild diploid, G. armourianum (D2–1). Development of the tri-species hybrids was summarized by Robinson et al. (2007). Here, we used two released LONREN lines (Bell et al., 2014), as well as crop science, vol. 56, july– august 2016
a number of other backcross-inbred lines that contain Renlon and differ in pedigree and/or alien segment constitution ( Table 1).
Classification of Resistant Lines Previously synthesized Renlon lines were categorized genetically according to recombination events relative to Renlon , the centromere, proximal and distal SSR markers, and the distal phenotypic marker Fzglon that dominantly confers “green fuzz” (Dighe et al., 2009), where “fuzz” refers to the short fibers of the seed maternal coat rather than “lint”, the long fibers. The Renlon -containing introgression lines were classified as containing the “whole alien segment” or a smaller (recombined) segment, each of the latter was further defined as “DCO” or “PCO”, according to the relative position of the prior recombination event, as described below. “DCO” resistance lines were identified as the product of a previous crossover distal to the Renlon gene because these lines contain proximal alien SSR markers BNL3279_114, BNL1066_156 and BNL836_215, but lack the distal alien marker CIR316_191 and phenotypic green-fuzz marker Fzglon (Fig. 2). Conversely, “PCO” resistance lines were identified as the product of a previous crossover proximal to the Renlon gene because these lines contain the product of a proximal crossover (between Renlon and the centromere), according to the presence of distal alien SSR marker CIR316_191 and phenotypic marker Fzglon , and the absence of the proximal SSR markers BNL3279_114, BNL1066_156 and BNL836_215 (Fig. 2). Lines with the “whole alien segment” were identified by retention of
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Fig. 2. Diagrammatic representation and categorization of distal crossover (DCO) or proximal crossover (PCO) recombination products characterized with the aid of simple sequence repeat (SSR) markers that closely flank the Renlon gene. Before recombination between these SSRs, the alien segment (red) is large and spans both SSRs. A nearby crossover can be detected by loss of an alien marker. Nearby DCOs lack CIR316_191 and nearby PCOs lack BNL3279_114.
all of the aforementioned proximal and distal markers (Fig. 2). PCO and DCO line classifications are shown in Table 1.
Leaf DNA Extraction Methods We used two DNA isolation procedures, conventional leafbased extraction and nondestructive seed-based extraction. Conventionally prepared DNA samples of the screening panel and parental reference genotypes from each population were extracted using young, folded or newly unfolded leaves by DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA yield was estimated using a NanoDrop2000 (Thermo Scientific, Waltham, MA) and the DNA diluted to 10 ng mL –1 for PCR amplification.
Seed DNA Extraction Methods DNA was extracted nondestructively from individual undelinted seed using the seed DNA extraction method of Zheng et al. (2015). We used three plates per set of 88 extractions, one of which was modified (Zheng et al., 2015). The last column of each plate was left open, as these positions were subsequently used for conventionally extracted reference controls. To facilitate embryo tissue sampling and seed archiving, seed were loaded into a modified PCR plate (88 seed per plate). One non-modified PCR plate served as the receptacle for collection of tissue from 88 tissue samples, as well as the respective DNA extractions, the two-fold dilutions and cryo-storage. The second non-modified 96-well plate was used to prepare working solutions; 10 mL of DNA extract (twice diluted) was transferred for each of the 88 seed-derived DNA samples, and then the DNA samples were diluted another 10-fold.
4
SSR and SNP Assays
Simple sequence repeat and SNP assays were run using 2 mL of diluted DNA (working solution) per assay; the last column of the assay plate was used for conventionally prepared (DNeasy Plant Mini Kit, Qiagen) parental DNA samples and two non-template controls. Unlabeled SSR primers were purchased from Integrated DNA Technologies, Coralville, IA. The PCR mixture (10 ml) used for SSR amplification contained ~10 ng of template DNA, 0.4 mM of each SSR primer (forward and reverse), 0.1 mM of dNTPs (Qiagen), 0.4 U of Taq polymerase (GenScript, Piscataway, NJ), and 1X PCR buffer made from the 10X buffer provided by the manufacture with a final concentration of 1.5 mM MgCl2. DNA was amplified for 38 cycles, each including 94°C for 30 sec, 56°C for 30 sec, and 72°C for 1 min, with initial denaturing of template DNA at 94°C for 5 min and a final extension at 72°C for 7 min. Amplified DNA samples were diluted by adding 5 mL of dilution buffer (Qiagen) and analyzed by an eGene capillary electrophoresis using a QIAxcel DNA High Resolution Kit (1200; Qiagen) or Advanced Analytics Fragment Analyzer (dsDNA reagent Kit, 35–500 bp [500 samples] DNF-900-K0500). Bands were scored visually from digital images generated by the respective software. For SNP KASP assays, plates were initially run for the recommended 38 cycles on the LGC genomics SNP line, centrifuged, and then read on the PHERAstar plate reader (BMG Labtech, Ortenberg, Germany). The PHERAstar files were imported into KlusterCaller (LGC Genomics, Beverly, MA) for genotype calling. When graphed SNP datapoints for seed-derived samples were not acceptably clustered for genotype calling, the plate was run through three more thermocycles and the plates were re-read, reimported and re-evaluated; this was repeated until scorable clusters were formed (Zheng et al., 2015).
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Table 2. Screening panel for Renlon –linked single nucleotide polymorphism markers. The different nematode resistance lines 7127, 7123, 7117, 7131, 3401, and 3402 were derived from the original HLA family. 7127†
7123†
7117‡
7131¶
3401§
3402§
7127
7123
7117
7131
340
3402
HLA††
G. longicalyx FM 966
G. armourianum NemX
HTC
HLA
G. longicalyx FM 966
G. armourianum NemX
HTC
† 7127 and 7123 bear different distal crossover events. ‡ 7117 bears a proximal crossover. ¶ 7131 bears a distal crossover (heterozygous). § 3401 and 3402 contain large alien segments, where 3401 is heterozygous. †† HLA is the tri-species hybrid, where G. longicalyx, FM 966, G. armourianum, and NemX are among the parents of HLA or its backcross derivatives.
Selection of Renlon –Linked SNP Markers By previous efforts, a total of 106,795 Gh–Gl SNPs were developed by RNA-seq and in silico filters and comparisons (Hulse-Kemp et al., 2014). SSR markers of the Renlon map (Dighe et al., 2009) and the Gh–Gl SNPs were aligned to the G. raimondii (D5) reference genome sequence (Paterson et al., 2012) using the Burrow–Wheeler Aligner in Galaxy (https:// usegalaxy.org) with its default settings. We eliminated class-III SNPs, those with one genome specific polymorphism (GSP) or indel within 50 bases of the SNP, repetitive SNPs, SNPs from the same RNA-seq contig, and lastly, SNPs for which primers could not be designed. The selected SNPs were validated with a screening panel that consisted of 11 genotypes, including parental controls and several different homozygous and heterozygous reniform nematode resistance lines (Table 2). ‘FM 966’ and ‘Acala NemX’ (NemX) were included as susceptible controls because they served previously as parents in the backcrossing program used to develop advanced reniform nematode resistant lines. Gossypium amourianum was included as it was used as a crossing “bridge” when creating the original HLA tri-species hybrid, and G. longicalyx provided the reniform nematode resistance gene, Renlon.
Validation of Ren –Linked SNP Markers lon
Allele-specific primers and allele-flanking primers for the selected SNPs were designed using the Batch Primer3 software program (You et al., 2008). Default settings were used to select both the SNP-specific and flanking primers with the only exception being maximum product size of 100 bp, minimum product size of 50 bp, and minimum and maximum primer melting temperatures of 55 and 63°C, respectively. Likewise, default settings were also used for penalty weights. Desirable SNP markers were validated against the screening panel using KASP assays (LGC Genomics). A six-by-four well format with two replicates of each genotype was used for screening Renlon –linked SNPs (Table 2).
BC1F1 for Mapping and Linkage Disruption
We developed large BC1F1 seed populations to address two objectives that are detailed below—high-resolution mapping and linkage disruption. To create the BC1F1 seed, we first created Renlon heterozygotes by crossing the homozygous PCO and DCO lines described above with G. hirsutum ‘FiberMax 966’
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(FM 966) in a greenhouse during spring of 2009. Field-grown F1 plants were backcrossed (testcrossed) to FM 966 (nematode susceptible) to construct the large BC1F1 populations during the summers of 2010 and 2011.
High-Resolution Mapping To create high-resolution maps of distal and proximal segments, seed DNA was extracted nondestructively (Zheng et al., 2015) from two subsets of 880 BC1F1 seeds, from one PCO-specific line (7117) and one DCO-specific line (7123) for a total of 1760 seed. SNPs suitable for the high-resolution mapping were chosen from among the validated Renlon –linked Gh–Gl SNPs, according to good clustering of KASP assays, as well as the distribution of their D5 -genome alignment sites. SNP genotyping was performed in 96-well plates using KASP assays. Genotype files were analyzed using JoinMap 4.1 (Kyazma, 2013) or maximum likelihood mapping with default settings to estimate the genetic distance of the genotyped SNP markers.
Populations and Marker-Assisted Selection for Linkage Disruption We randomly selected a 10-fold larger set of BC1F1 seeds (17,600) from the 2011 summer backcrosses described previously. After physically organizing them, we nondestructively sampled each of them for DNA extraction by the methods of Zheng et al. (2015). Using the extracts, the two closest SNPs on opposite sides of SSR marker BNL1231, Gl_168758 and Gl_072641, were used for large-scale screening to identify recombinants with breakpoints very close to Renlon. Two hundred 96-well plates of 88 seed DNA samples, seven conventionally prepared DNAs from parental controls and one non-template control per plate were screened using the two Renlon -flanking SNP markers. The parental controls included 7117, 7123, 7131, G. longicalyx, FM 966, G. armourianum and HLA. The KASP assay (http:// www.lgcgenomics.com/) was used for SNP amplification.
Homologous vs. Homeologous Recombination We created small seed populations that would allow us to compare recombination rates across a small region of interest when it is entirely heterozygous versus at least partially homozygous (Fig. 3). When the alien region is entirely heterozygous, meaningful crossover events must occur between homeologous (A–F) segments, whereas when the region is at least partially homozygous (e.g., F–F), crossover events could occur between homologous alien segments, for which the rates would expectedly be higher. To create the germplasm for estimating the homeologous recombination rates near Renlon , we backcrossed line 21-2, which contains the whole alien segment, with G. hirsutum ‘Deltapine 5415’, and then testcrossed the resulting F1 hybrid Renlon heterozygotes to G. hirsutum ‘Deltapine 5415’ to produce testcross seed (Fig. 3 “BACKCROSS”). To estimate homologous recombination rates, we intercrossed previously derived PCO and DCO lines with overlapping common segments, namely PCO line 7117 and DCO line 7123, then testcrossed the PCO × DCO F1 hybrid to the cultivar FM 966 (Fig. 3 “INTERCROSS”). To detect recombination events in the vicinity of Renlon , we used two codominant SNPs that had been previously mapped
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Fig. 3. Recombination rate comparison between BACKCROSS and INTERCROSS methods by molecular marker analysis. D refers to a distal marker locus, and P refers to a proximal marker locus. For the BACKCROSS method, recombinants were sought in regions A or B. For the INTERCROSS method, recombinants were sought between preexisting distal crossover (DCO) and proximal crossover (PCO) recombinants. Marker-assisted selection (MAS) was used to recover recombinants between flanking marker loci D and P, some of which should occur in the common region from G. longicalyx (green shading).
to opposite ends of the common alien region between the PCO and DCO lines used herein as parents of the INTERCROSS F1 (see below and Fig. 3). The SNP Gl_117570 is proximal to Renlon and referred to hereafter as locus “P”, whereas the SNP Gl_212476 is distal to Renlon and referred to hereafter as locus “D”. We nondestructively extracted DNA from 88 BACKCROSS and 88 INTERCROSS seeds. The two sets of 88 DNA extracts were placed into separate 96-well PCR plates, along with the previously described panel of seven conventionally prepared DNAs from parental clones and lines, and one non-template control. All samples were tested using KASP assays for the two SNP markers. The Renlon parent line (21-2) used to produce the BACKCROSS F1 hybrid is homozygous for a very large alien segment that spans SSRs BNL836_215 and CIR316_191. For the BACKCROSS method depicted in Fig. 3, G. longicalyx alleles (denoted by superscript “lon”) from the original LONREN line with the whole alien segment can be amplified by both SNP markers Gl_117570 (P) and Gl_212476 (D), Plon Dlon//PhirDhir. Given testcrosses of the BC1F1 with G. hirsutum ‘Deltpine5415’, each reciprocal homeologous recombination event in the BC1F1 generation will result in testcross progeny with one of the two 6
SNPs being homozygous for G. hirsutum alleles (superscript “hir”), PhirDlon//PhirDhir or Plon Dhir//PhirDhir. Otherwise, each non-recombinant testcross progeny will have a parental genotype, either Plon Dlon//PhirDhir or PhirDhir//PhirDhir (Fig. 3). The two Renlon parent lines (7123 and 7117) used to produce the INTERCROSS F1 hybrid are each homozygous for a Renlon bearing segment truncated by nearby prior recombination (DCO or PCO); the F1 possesses a relatively small “common region” (homozygous for G. longicalyx alleles). As depicted in Fig. 3, the INTERCROSS F1 from line 7117 × line 7123 has the expected SNP genotype Plon Dhir//PhirDlon. After testcrossing with G. hirsutum ‘FiberMax966’, homologous recombination in the segment of interest would lead to recombinant genotypes PhirDhir//PhirDhir or PlonDlon//PhirDhir. If there is no recombination, then the progeny genotypes will remain heterozygous for parental SNP combinations, either DCO PlonDhir//PhirDhir, or PCO PhirDlon//PhirDhir (Fig. 3). The genotypic frequencies were compared using Pearson’s chi-square test (c2; Pearson, 1900).
Phenotypic Screening For evaluation, seed were produced by self-pollination from recombinant plants identified by MAS. The expected ratio
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among selfed seeds from a new recombination product would be 1:2:1 for the new allele combination. The seed were collected, ginned, delinted and stored in an incubator at 40°C for 2 wk to increase consistency of germination. Before phenotypic screening, a set of 10 selfed seeds from each of the selected recombinants was planted, and DNA was extracted using young, folded or newly unfolded leaves from the seedlings by the DNeasy Plant Mini Kit (Qiagen). The DNA was then tested for BNL1231_null. To date, BNL1231_null is the marker closest to the Renlon gene, and as far as is known, it has co-segregated 100% with both reniform nematode resistance and seedling stunting so far. Selfed seeds from selected recombinant plants were used to test for stunting and reniform nematode resistance, and thereby to determine if linkage was broken between Renlon and nearby loci that we hypothesize could control susceptibility to stunting.
Stunting Test Assay In the stunting assay, FM 966 served as a nematode-susceptible stunt-resistant control, and the experimental line LONREN MR-19 served as the nematode-resistant stunt-susceptible control. Five experimental replicates were used for each control. Ten BC1S1 progeny from each recombinant were used because they were expected to segregate 3:1 for susceptibility to stunting. The stunting assay experiments were conducted in growth chambers at the USDA-ARS, College Station, Texas. The temperature was set to 20°C during night (11 h) and 25°C during daytime (13 h). A fine sandy loam topsoil was bought from a local dealer (Oppie’s Topsoil, Sand, and Gravel, Bryan, TX), and mixed 3:1 with washed sand. The wetted mixture was pasteurized with aerated steam treatment at 75°C for 16 h on each of two consecutive days. Pasteurized soil was equilibrated in growth chambers for at least 24 h before use. Planting cups, drilled and fitted with a fiberglass screen for drainage, were filled with about 500 g of sandy loam soil. One day before planting seedlings, a 50 g core of soil (5 cm length, 2.5 cm diam.) was replaced in the center of the cup with stunting assay soil, which was sandy loam soil plus natural mixture of fungi, including Thielaviopsis basicola and Fusarium spp., specifically FOV Race 1, and reniform nematodes, and previously was shown to cause severe stunting of LONREN line MR-19. The core soil mix for the stunting test contained 43.5 nematodes per gram of soil. The nematode population was originally from Arkansas (provided by R.T. Robbins, University of Arkansas, 2006). Seeds were scarified, rinsed with hot tap water (50°C) for 20 s, rolled in germination paper (No. 28334-194, Andwin Scientific, Schaumberg, IL), and germinated at 30°C for 24 h, and then at 14°C for another 24 h, to obtain radicles of consistent length (~5 cm). Uniform seedlings were transplanted individually into cups. Each cup was fertilized before planting by applying 50 mg of 10–30–20 fertilizer (Scotts Peters 99350 Water Soluble Peat Lite Plant Starter Fertilizer) in 50 mL water. After 1 wk, 25 mg of 10–30–20 fertilizer in 50 mL water was added to each cup; at subsequent weekly intervals until harvest, each cup was fertilized with 50 mg of 15–5–25 fertilizer (Scotts Peters 99220 Water Soluble Peat Lite Flowering Fertilizer) in 50 mL water. Plant height data were recorded 2 wk after planting.
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Reniform Nematode Resistance Test Assay In the reniform nematode resistance test assay, FM 966 was used as a nematode-susceptible control, and experimental line MR-19 as a nematode-resistant control. Eight experimental replicates were used for each control. Four replicates of LONREN-2, a resistant germplasm line, were also included in the test. Comparisons to the susceptible and resistant controls allowed assessment of BC1S2 seedlings from homozygous BC1S1 plants that had been selected previously by MAS from five recombinant BC1F1 plants. Segregation for the SNPs was not expected among the BC1S2 seedlings of a given family. The experiments were conducted in a growth chamber at the USDA-ARS, College Station, Texas. The temperature was set at 28°C for 13 h of light and 22°C for 11 h of darkness. The soil mix used for nematode assays was made by mixing 90.9 L of sandy loam, 7.6 L of vermiculite, 600 mL of dolomite, and 300 mL of gypsum. The soil was pasteurized and equilibrated. Seed germination and transplanting steps were the same as for the stunting test assay. Reniform nematodes were extracted by the Baermann funnel technique (Robinson and Heald, 1991) from the commercial sandy loam soil infested with an Arkansas population of reniform nematode provided by R.T. Robbins. Reniform nematodes were injected into the soil surrounding the seedlings using a 1 mL syringe (B-D Luer Lock Part No. 309628). Approximately 4000 reniform nematodes were injected for each cup. Three cores of soil from each cup were sampled 8 wk after inoculation, and extracted by the Baermann funnel technique. Then nematodes were counted and counts per gram of soil were calculated. Statistical analyses were performed using SAS v.9.3 (SAS Institute, 2011).
Results and Discussion Large-Scale Seed Genotyping The methods of Zheng et al. (2015) enabled the extraction of large numbers of DNA samples from individual genotypes (~20,000) with reduced labor and little cost and was a key factor enabling this study. With comparable reductions in marker costs, it will become very feasible to use marker-assisted technologies extensively for cotton germplasm introgression, genetic dissection, analysis, and selection. The ability to extract DNA nondestructively from seed allowed for greater temporal flexibility, and the MAS with SNPs greatly reduced the numbers of seed that had to be planted. These kinds of MAS based on targeted SNP activities seem certain to become increasingly common in cotton breeding and related research, given recent strides in SNP discovery (Ashrafi et al., 2015; Hulse-Kemp et al., 2014, 2015a; Islam et al., 2015; Wang and Chen et al., 2015), development of a high-quality high-density SNP array (Hulse-Kemp et al., 2015b), some progress in validated genotyping by sequencing (Gore et al., 2014), G. hirsutum genome sequence assembly (Li et al., 2015; Zhang et al., 2015), and large-scale genomewide SNP mapping (Hulse-Kemp et al., 2015b; Wang
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et al., 2015). The ability to conduct large-scale seedbased MAS will revolutionize the science, scope and efficacy of wide-cross cotton breeding; these trends will foster the assimilation of much more germplasm diversity into mainstream cotton breeding. Desirable genes of interspecific origin are likely to be linked to genes with undesirable effects, especially when linkages are intensified and extended by reduced genomic homology. MAS will foreseeably enable widespread applications in quantitative trait locus dissection and derivation of downstream introgression products that contain beneficial alien genes but not linked deleterious ones. This increased capability will foster increased use of wide-cross germplasm introgression as part of breeding programs dedicated to deriving elite genotypes.
Renlon –Linked SNPs Alignment of Renlon –linked SSR marker sequences to the D5 genome assembly (v2.1) identified the corresponding region of Scaffold_7 (chromosome 7; Table 3). From the initial set of 106,795 putative SNPs, about 700 of Gh–Gl SNPs were aligned to this 9 Mbp region, which equates to a density of about one SNP per 13 kbp. From this region, 126 Gh–Gl in silico SNPs were selected and KASP primer sets were designed. Based on testing of the KASP assays against the screening panel (Table 2), 85 Gh–Gl SNPs were validated as functional assays for loci linked to the Renlon gene (SNP sequences and primers are in the Supplemental Material). The overall rate at which in silico SNPs between G. longicalyx and G. hirsutum genomes yielded well-clustered SNP assays (KASP) was over 90% (unpublished result), whereas the rate at which we converted the selected Gh–Gl SNPs to KASP was 85 of 126 (~67%), and reflected selection for robust amplification and linkage associations, based on results from the screening panel. Based on results from the screening panel, the 85 SNPs were assigned to the proximal (47), distal (36) and “common” (11) regions, as defined by the segments in lines 7117 and 7123. The 10 Gh–Gl SNPs chosen for mapping of the 7117 (PCO) family aligned to distal positions 56,835,707 to 60,340,986 bp (~3.5 Mbp), in Chr. 07 of the D5 assembly, whereas the 10 Gh–Gl SNPs chosen to analyze the 7123 (DCO) family aligned to D5 Chr. 07 from proximal positions 52,275,364 to 57,279,176 bp (~5 Mbp), indicating that their respective alien segments share a common region extending at least from 56,835,707 to 57,279,176 bp (~400 kbp) in the D5 assembly. Two SNPs in the common region between lines 7117 and 7123 were selected for mapping and MAS, namely Gl_168758 and Gl_072641; according to the D5 alignment results, these SNPs define a ~400 kbp segment in the D5 genome and flank the SSR marker BNL1231 on opposite sides. In addition, 16 more SNPs were selected across the proximal and distal regions (8 each), such that they were distributed 8
Table 3. Mapped simple sequence repeat (SSR) markers aligned to the D 5 _v2.1 assembly. Mapped SSR markers
Scaffold or chromosome
Position
CIR316
07
59563000
BNL1231
07
57124927
BNL3279
07
56142974
BNL1066
07
54550465
BNL836
07
52098818
across the corresponding D5 reference genome sequence assembly regions, which are about 3.5 and 5 Mbp, respectively. Given that the Gossypium F and A genomes are larger than the D5 genome (Hendrix and Stewart, 2005), it is reasonable to infer that the size of the common region in the D5 alignments (~400 kbp) likely constitutes a minimal estimate of the size of the common region between the alien F-genome DCO and PCO segments. The 18 selected Gh–Gl SNP sequences and KASP assay primers are highlighted in the Supplemental Material.
High-Resolution Mapping Two populations of 880 testcross seed each from DCOand PCO-heterozygous parents were evaluated to identify recombinants and map SNPs in proximal, common and distal segments. Results from the DCO and PCO families differed markedly. Whereas the 7123 (DCO) testcrosses included 7 recombination events, the 7117 (PCO) testcrosses included none (Table 4). Because the numbers of SNP-identified recombination events were unexpectedly disparate, we ran flanking codominant SSR markers located proximally (MUSB0404) and distally (MGHES016) on each of the respective seed DNA samples to detect any single crossover products, and we ran all of the KASP assays again for the seven recombinants. The results confirmed the initial results, so the 10 Gh–Gl SNPs scored in the 7123 (DCO) family were mapped by linkage analysis (Table 5). While not all 10 SNPs were uniquely binned, a comparison of linkage mapping results to the order deduced by alignment to the D5 genome showed them to be congruent. The seven recombinant seed (Table 4) were germinated to produce plants and self-seed. The results confirmed inferences that SNPs Gl_168758 and Gl_072641 are in the common region and very closely linked. Among the 1760 seed, only one recombination event (D1F3) occurred in the common region, which equates to 0.06 cM overall, or 0.12 cM for the DCO family alone (Table 5). Given that these two SNPs putatively flank BNL1231 and Renlon , their strong linkage in this germplasm indicated that they could be used for large-scale MAS of products from homologous recombination in the common region.
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Table 4. Single nucleotide polymorphism marker genotypes indicating locations of recombination events in BC1F1 seed from family 7123. 7123 family
Gl_178356 Gl_117394 Gl_085251 Gl_200380 Gl_187401
Gl_142101 Gl_012150
Gl_117570
Gl_168758 Gl_072641
D1F3
H†
H
H
H
H
H
H
H
H
A
D1H4
H
H
H
H
H
A
A
A
A
A
D2C10
H
H
A
A
A
A
A
A
A
A
D3E4
H
H
H
A
A
A
A
A
A
A
D3H1
A
A
A
A
H
H
H
H
H
H
D6F4
A
A
A
A
A
A
A
A
H
H
D8A11
A
A
A
A
A
A
A
A
H
H
† A, homozygous for G. hirsutum allele; H, heterozygous for G. longicalyx allele.
Table 5. High-resolution mapping results of Ren lon –linked single nucleotide polymorphism (SNP). SNPs in proximal region
SNPs in distal region
Position on D5_v2.1
Map position based on DCO-heterozygote†
52275364
0.802
Map position based on PCO-heterozygote‡
-––––––––––––––––– cM -–––––––––––––––– Gl_178356
–
Gl_117394
52513762
0.802
–
Gl_085251
52847958
0.688
–
Gl_200380
53688922
0.574
–
Gl_187401
54605631
0.460
–
Gl_142101
55188861
0.347
–
Gl_012150
55404836
0.347
–
Gl_117570
55971263
0.347
–
56835707
0.117
0
Gl_072641
57279176
0
0
Gl_212476
57435262
–
0
Gl_052088
57775963
–
0
Gl_148225
57834425
–
0
Gl_104307
58465267
–
0
Gl_211386
58627706
–
0
Gl_168758
Gl_168758
Gl_072641
Gl_199350
59030131
–
0
Gl_208281
59481497
–
0
Gl_082005
60340986
–
0
† DCO, distal crossover. ‡ PCO, proximal crossover.
Marker-Assisted Selection for Linkage Disruption Testcross seed from F1 hybrids heterozygous for previously characterized PCO and DCO alien segments (Table 1) were screened to find recombination products near Renlon. Using nondestructive seed-based MAS with the two closest common-region SNPs, Gl_168758 and Gl_072641, five new recombinants (Table 6) were tentatively identified among 17,600 testcross seed, of which 17,215 (~98%) were successfully genotyped. To verify seed-DNA SNP results, the putatively recombinant seed were germinated and grown to produce self-seed. DNA extracted from each of ten self-seed per putative recombinant was tested against BNL1231, and after germination, DNA extracted from young leaves was re-tested against BNL1231. By crop science, vol. 56, july– august 2016
scoring for segregation among the self-seed, two of the five recombinant lines, D1F3 and LS33C5, were shown to be homozygous for BNL1231, and three of the recombinant seedlings, LS53F11, LS78D5 and LS149B11, were found to segregate for BNL1231 and BNL1231_null. The SSR results from seed extractions were confirmed using higher quality DNA from young leaves. According to previously established linkage relationships, the families homozygous for BNL1231 would be found to be reniform susceptible and not susceptible to stunting, while those segregating BNL1231 and BNL1231_null would co-segregate for reniform resistance and stunting. Phenotypic tests were performed next to determine if any of the recombinants had resistance to both reniform nematode and stunting.
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Table 6. Marker genotypes for the five new MAS-selected recombinants near Renlon. Recombinant progeny
DCO donor
Gl_168758
BNL1231
Gl_072641
New break point
D1F3
7123
H†
A
A
Proximal to BNL1231
LS33C5
7112
H
A
A
Proximal to BNL1231
LS53F11
7110
A
H
H
Distal to BNL1231
LS78D5
7126
A
H
H
Distal to BNL1231
LS149B11
7110
A
H
H
Distal to BNL1231
† A, homozygous for G. hirsutum allele; H, heterozygous for G. longicalyx allele.
Table 7. Seedling height 2 wk after planting. Genotypes (BC1F1 lines or families)† Plant
FM 966
MR-19
D1F3
LS33C5
LS53F11
LS78D5
LS149B11
1‡ 2 3 4 5
––––––––––––––––––––––––––––––––––––––––––––––––––– cm ––––––––––––––––––––––––––––––––––––––––––––––––––– 8.4§ 3.7 9.1 6.3 6 4.1 8.1 6.3 3.1 6.4 8.8 8.3 7.8 3.8 5.8 3 8.5 8.7 6 7.1 4.2 7.8 3.2 7.3 7.3 4.4 5.2 9.3 5.5 5.4 7.8 7.6 6.5 6.1 4.2
6 7 8 9 10 Mean SD SE
na na na na na 6.76 1.27 0.57
na na na na na 3.68 1 0.45
6.2 6 7.7 7.7 6.3 7.3 1 0.30
9.8 9.6 8.7 9.6 6.7 8.31 1.19 0.38
6.3 3.6 4.9 5.5 4.1 5.56 1.31 0.414
5.5 7.7 4.1 5.2 4 5.68 1.38 0.436
5.4 9.2 4.2 4.4 3.7 5.65 2.17 0.686
† Two genotypes were used as controls, “non-stunting control” FM966, and “stunting control” MR-19. BC1S1 plants from five families, D1F3, LS33C5, LS53F11, LS78D5 and LS149B11 were tested for stunting. ‡ 5 seedlings tested for the control genotypes; 10 seedlings tested for each of the BC1S1 families. § Seedling height measured after 2 wk of transplanting. Segregations were observed in the stunting families, some seedlings were as short as MR-19, some seedlings were as tall as FM966. The non-stunting families were not segregated and performed similarly as FM966. Color orders denote individuals with similar heights.
Stunting Tests BC1S1 progeny produced by each of the SNP-selected recombinants were evaluated for stunting by assessing seedling heights at 2 wk after planting (Table 7). Because the reliability of the single-plant stunting assays is very high, we were able to use BC1S1 progeny evaluations to determine if any of the recombinant lines were segregating for the stunting trait. The self-progeny from D1F3 and LS33C5 did not show stunting, whereas those from LS53F11, LS78D5 and LS149B11 segregated for stunting (Table 7). The results indicated that BNL1231_null remained linked with stunting. A reniform resistance test was then run to determine whether BNL1231 was uncoupled from the Renlon gene. Reniform Nematode Resistance Tests To evaluate resistance to reniform nematode, we avoided unreplicated assays of segregating BC1S1 seedlings, because single-plant reniform nematode assays are imperfect indicators (Robinson et al., 2007). We instead used MAS with SNPs to identify and select BC1S1 plants that were homozygous for the newly recombinant segments of the common region, then grew them to obtain BC1S2 10
seeds, each family of which was uniformly homozygous for the respective recombination product. The BC1S2 seed were harvested and used for replicated assay-based testing. The numbers of reniform nematodes per gram of soil were determined and evaluated for recombinant BC1S2 lines that were homozygous for the recombination products. The “resistant controls”, MR-19 and LONREN-2, had significantly fewer nematodes per gram of soil than the “susceptible control”, FM966 (Fig. 4). The BC1S2 plants from LS53F11, LS78D5 and LS149B11 had extremely low numbers of reniform nematodes, similar to the LONREN lines. The BC1S2 plants from D1F3 and LS33C5 had very high numbers of reniform nematode, similar to FM 966. The results indicated that BNL1231_ null remained linked with the resistance, and that the resistance and stunting were not uncoupled from each other, in spite of intense selection.
Implications for Stunting Mechanism The absence of a recombinant with stunt-free reniform nematode resistance could be due to inadequate population size, especially given the depressed rates of homeologous recombination in the targeted PCO and DCO regions.
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Fig. 4. Boxplot displaying mean number of nematodes (Ne) per gram of soil for each genotype at 8 wk after inoculation. The horizontal axis shows the genotypes tested in the assay. MR-19 and LONREN-2 are the resistance controls, FM966 is susceptible control. BC1S2 plants from five families LS53F11, LS78D5, LS149B11, LS33C5 and D1F3 were tested.
However, other possible explanations exist. Stunting might be due to pleiotropy, primary or secondary effects of the nematode resistance (Sikkens et al., 2011, Zheng et al., 2013). We have not excluded the possibility that the hypersensitive reaction itself is responsible directly and/or indirectly for the stunting. Under heavy reniform nematode pressure, the plant roots may continue to kill cells in the vicinity of any attempt by the nematode to feed, and these may create multiple lesions that collectively impede the normal functioning of the roots and/or open numerous portals for secondary infections. In this scenario the high efficiency of the resistance mechanism to halt nematode infection is the plant’s own undoing. Whereas the Renlon mechanism works well under light to moderate nematode pressure, e.g., following a grass rotation crop, in fields with high initial populations, the mode of LONREN resistance is too damaging to the plant itself to permit healthy growth, such that the plants exhibit high resistance to reniform nematode, but poor tolerance. However, the situation is complicated by a growing body of evidence that LONREN lines are more susceptible to certain soil fungi in the presence of the nematode (Bell et al., 2011, 2012; Zheng et al., 2013), and that such susceptibility significantly accentuates seedling stunting. The cause of the increased susceptibility to fungi is yet to be established; it could be due to linked gene(s), pleiotropic genetic effects and/or secondary effects linked to cell necrosis in the vicinity of reniform feeding sites.
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Implications for Recombination The linkage disruption effort led to just five recombination products among 17,600 seed, a frequency of just 0.0003. Considering the G. hirsutum genome is about 4000 cM (Hulse-Kemp et al., 2015b) and 2.5 Gbp (Hendrix and Stewart, 2005), its average rate of recombination is about 1.5 cM per Mbp. Given that we expect the alien F1 segment to be at least as large as the equivalent D5 segment (~0.4 Mbp), the observed rate of recombination is about 1/20th that of the average homologous G. hirsutum recombination rate. We also observed that the recombination rates between G. longicalyx and G. hirsutum segments were significantly depressed in the high-resolution mapping experiment, which involved 1760 seed. Depressed rates of homeologous recombination (10% or less) were also indicated by previous whole-chromosome meiotic recombination analyses (Robinson et al., 2007) and in the previous large-segment genetic maps between G. longicalyx and G. hirsutum (Dighe et al., 2009), compared to [AD]1–[AD]2 interspecific c11 recombination rates between G. barbadense and G. hirsutum (Yu et al., 2012; Hulse-Kemp et al., 2015b). Thus, evidence at the whole-chromosome, large-segment and local levels indicates homeologous (F–A) recombination rates are lower than 1/10 and probably less than 1/20 the normal homologous AD–AD rate.
F–F versus A–F Recombination Using SNPs Gl_117570 and Gl_212476 to screen for recombinants near Renlon in identically sized (88) INTERCROSS
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Table 8. Number of recombinants found by marker analysis in the testcross progenies derived from INTERCROSS method and BACKCROSS method. Type of cross method INTERCROSS Recombinant Non-recombinant Total
22 (11.5)† 66 (76.5) 88
BACKCROSS 1 (11.5) 87 (76.5) 88
Total 23 153 176
† The observed and expected (shown in parentheses) numbers of recombinant and non-recombinant seed.
and BACKCROSS testcross seed populations, we characterized homologous versus homeologous recombination rates in the immediate vicinity of Renlon. These two codominant SNP loci mapped closely (
