Molecular mapping and characterization of QBp.caas-3BL for black point resistance in wheat (Triticum aestivum L.)

We fine-mapped QBp.caas-3BL for black point resistance in an interval of 1.7 Mb containing five high-confidence annotated genes and developed a KASP marker suitable for selection of QBp.caas-3BL. Wheat black point, which occurs in most wheat-growing regions of the world, is detrimental to grain appearance, processing and nutrient quality. Mining and characterization of genetic loci for black point resistance are helpful for breeding resistant wheat cultivars. We previously identified a major QTL QBp.caas-3BL in a recombinant inbred line (RIL) population of Linmai 2/Zhong 892 across five environments. Here we confirmed the QTL in two additional environments. The genetic region of QBp.caas-3BL was enriched with newly developed markers. Using four sets of near isogenic lines, QBp.caas-3BL was narrowed down to a physical interval of approximately 1.7 Mb, including five annotated genes according to IWGSC reference genome. TraesCS3B02G404300, TraesCS3B02G404600 and TraesCS3B02G404700 were predicted as candidate genes based on the analyses of sequence polymorphisms and differential expression. We also converted a SNP of TraesCS3B02G404700 into a breeding-applicable KASP marker and verified its efficacy for marker-assisted breeding in a panel of germplasm. The findings not only lay a foundation for map-based cloning of QBp.caas-3BL but also provide a useful marker for selection of resistant cultivars genotypes in wheat breeding.


Introduction
Wheat is an important staple crop that provides about 20% of calories for humankind. Black point (also known as kernel smudge) is a severe disease characterized by dark discoloration at the embryo end of wheat grains and occurs in most wheatgrowing regions of the world. Black point does not typically cause yield loss, but reduces grain quality. It causes inferior grain appearance, processing and nutrient quality as well as lower seed vigor (Liu et al. 2016). The causes of black point are complicated, including biotic and abiotic causal agents. It can be caused by several pathogens, such as Alternaria alternata (Southwell et al. 1980;Perelló et al. 2008; Davis and Jackson 2009;Busman et al. 2012), Bipolaris sorokiniana (Kumar et al. 2002;Li et al. 2020) and Fusarium proliferatum (Conner et al. 1992;Desjardins et al. 2007;Busman et al. 2012). It was also reported that the formation of black point was due to an enzymatic browning reaction caused by peroxidases (POD) (March et al. 2007;Fernandez et al. 2014), polyphenol oxidase (PPO) (Fuerst et al. 2014) and lipoxygenase (LOX) (Porta and Rocha-Sosa 2002), which can transform phenolic compounds into brown or black pigments like melanins (Fuerst et al. 2014) and quinines (Tomás-Barberán and Espín 2001;Fernandez and Conner 2011). Some environmental factors, such as temperature and humidity, also have Communicated by Ian Mackay.
In our previous study, nine QTL for black point resistance were mapped on chromosomes 2AL, 2BL, 3AL, 3BL, 5AS, 6A, 7AL (2) and 7BS in a recombinant inbred line (RIL) population derived from the Linmai 2/Zhong 892 cross (Liu et al. 2016). QBp.caas-3BL was detected across five environments and explained 6.0-12.2% of the phenotypic variances, suggesting that it was a stable major QTL for black point resistance. Here QBp.caas-3BL was confirmed in another two environments. We created a series of near isogenic lines to fine-map QBp.caas-3BL and predicted its candidate genes. A cost-effective KASP marker was also developed to improve black point resistance in wheat breeding.

Plant materials
To confirm QBp.caas-3BL, we planted the Linmai 2/Zhong 892 RIL population in Anyang (Henan province) and Suixi (Anhui province) during the 2018-2019 cropping season. The residual heterozygous recombinant plants for QBp.caas-3BL were screened from the RIL population. Four sets of near isogenic lines (NILs) for QBp.caas-3BL were identified from the residual heterozygous recombinant plants and used for fine mapping (Table S1). The NILs were grown in single row plots of 2 m with approximately 40 plants per row and 20 cm between rows at Anyang during the 2018-2019 cropping season and Xinxiang (Henan province) during the 2019-2020 cropping season. A panel of 166 wheat cultivars from the Huang-Huai Wheat Region was used to validate the genetic effect of QBp.caas-3BL on black point response (Liu et al. 2017).

Phenotypic evaluation for black point
Black point in mature grains was assessed manually at least 15 days after harvest. Each RIL or NIL was scored for black point response (obvious discoloration around the embryos) by counting the number of black point-affected grains among three samples of 200. The percentage of black point-affected kernels was taken as the black point score for each plot. To ensure the accuracy of phenotyping, black point of all materials was scored by the same person under similar illumination conditions.

PCR-based genotyping by sequencing
To enrich the target region of QBp.caas-3BL, annotated genes were identified in the physical interval according to the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.1 (http:// plants. ensem bl. org/ index. html). The sequences from some annotated genes were retrieved and aligned to design gene-specific primers using DNAMAN software. The polymorphic sites between parents Linmai 2 and Zhong 892 were used to develop markers for fine mapping of QBp.caas-3BL. PCR was performed in reaction mixtures of 15 μl containing 7.5 μl of 2 × Taq PCR Mix (Beijing HT-biotech Co., Ltd., Beijing), 5 pmol of each primer and 50-100 ng of genomic DNA using an ABI Applied Biosystems Veriti 96 Well Thermal Cycler (Gene Co., Ltd., Shanghai). PCR amplification was performed at 95 °C for 3 min, followed by 35 cycles (94 °C for 30 s, 59-61 °C for 30 s and 72 °C for 1 min) and a final extension at 72 °C for 7 min. Primer pairs and their annealing temperatures for PCR are listed in Table S2. PCR products were recovered from 1.5% agarose gels and purified by TIANgel MIDI Purification Kit (Tiangen, Biotechnology Co., Ltd., Beijing). The sequencing of target PCR products was performed by BGI Genomics Co., Ltd in Beijing.

Development of a KASP marker
PolyMarker software (http:// polym arker. tgac. ac. uk/) was used to design KASP markers for target polymorphic sites (Table S3). The PCR cycling conditions were 94 °C for 15 min, followed by 10 touchdown cycles (94 °C for 20 s; 62 °C to 55 °C for 60 s), 26 cycles of 94 °C for 20 s and 55 °C for 60 s, 6 cycles of 94 °C for 20 s and 57 °C for 60 s, and one final acquisition cycle at 16 °C for 60 s (Collins et al. 2018). The KASP platform of LGC Co., Ltd was used to carry out genotyping and statistical analyses.

Quantitative PCR
Grains of Linmai2 and Zhong892 were sampled 25 days after flowering and used to isolate total RNA with a Total RNA Extraction Kit (Beijing TransGen Biotechnology, Cat#ER301-01). cDNA was generated with a PrimeScript RT Reagent Kit plus gDNA Eraser (TaKaRa, Dalian). Quantitative PCR (qPCR) was performed on cDNA samples produced from three biological replicates in a BioRad CFX system using iTaq Universal SYBR Green Supermix (BioRad, Beijing). The primer pairs for gene-specific qPCR were designed according to the gene sequences from RefSeq v1.1 (Table S4). The profile of qPCR was as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 59 °C for 20 s. A final dissociation stage was run to generate a melting curve for judgment of amplification specificity. Relative gene expression was normalized to elongation factor 1 alpha (EF1α) using the 2 −ΔΔCt equation.

Linkage mapping and QTL analysis
A genetic linkage map for QBp.caas-3BL was constructed using JoinMap 4.0 (http:// www. kyazma. com). QTL mapping was conducted by inclusive composite interval mapping (ICIM) in IciMapping 4.0 software (https:// www. isbre eding. net) with a LOD threshold of 3.0. The means of phenotypic data for RILs in each environment were used for QTL analysis.

Statistical analysis
Association analysis for declaration of significance at P < 0.05 in KASP marker validation was conducted by student's t-tests in Excel. The Excel software was also used to calculate the means, standard deviation and coefficients of variation in phenotypic data.

Confirmation of QBp.caas-3BL
In the previous study, QBp.caas-3BL was identified as a major QTL for black point in the RIL population of Linmai 2/Zhong 892 (Liu et al. 2016). To confirm the presence of QBp.caas-3BL, we re-investigated black point response of the RIL population in two additional environments (2019 Anyang and 2019 Suixi). Based on the phenotypes in these two environments, a QTL for black point was detected with a consistent genetic location of QBp.caas-3BL ( Fig. 1A; Liu et al. 2016) and explained 6.6-12.4% of the phenotypic variance. These results confirmed that QBp.caas-3BL is a stable QTL for black point.

Generation of secondary mapping populations for QBp.caas-3BL
To narrow down the target interval of QBp.caas-3BL, we created secondary mapping populations. Based on the genotyping data by the wheat 90 K SNP chip, four RILs with residual heterozygosity in the QBp.caas-3BL target region were identified using markers. We genotyped approximately 2000 individual plants from the heterozygous recombinant lines using the flanking markers RAC875_rep_c105184_88 (M1) and Tdurum_contig67350_494 (M12) (Fig. 1A; Table S2). Each of the so-called residual heterozygous recombinant lines had homozygous plants with contrasting genotypes in the target region of QBp.caas-3BL. Since the plants from each residual heterozygous recombinant line harbored contrasting genotypes in the target region but were homozygous for most other regions of the genome based on SNP arrays, they were treated as NILs of QBp.caas-3BL. Four sets of QBp.caas-3BL NILs were selected and used to create secondary populations for fine mapping (Table S1).

Prediction of candidate genes
Among the five candidate genes, TraesCS3B02G404300, TraesCS3B02G404400, TraesCS3B02G404500 and TraesCS3B02G404600 are bZIP family transcription factors that are orthologous to ABSCISIC ACID-INSENSITIVE 5 (ABI5) (Fig. 1C and S1), whereas TraesCS3B02G404700 is a homolog of protein phosphatase (Fig. S2). To determine the candidate gene(s) in QBp.caas-3BL, we compared the open reading frames (ORFs) of the five genes from parents Linmai 2 and Zhong 892 by sequencing. The ORFs of TraesCS3B02G404600 and TraesCS3B02G404700 contained sites polymorphic between Linmai 2 and Zhong 892. The SNP in TraesCS3B02G404600 was a synonymous mutation, whereas that of TraesCS3B02G404700 was a nonsynonymous mutation causing a serine-to-alanine substitution ( Fig. 2A  and 2B). We also investigated the expression patterns of the five genes in Wheat Expression Browser (http:// wheat-expre ssion. com/) and found that all were expressed in grains, suggesting that they could be involved in black point response (Fig. S3-7). All four ABI5 homologs, TraesCS3B02G404300, TraesCS3B02G404400, TraesCS3B02G404500 and TraesCS3B02G404600, have spike-and seed-specific expression patterns, whereas TraesCS3B02G404700 is highly expressed in all tissues (Fig. S3-7). We further compared the transcriptional levels of the five genes in developing grains using qPCR assays and observed that TraesCS3B02G404300,  Table S2; P1 and P2 represent Zhong 892 and Linmai 2, respectively; BPR, black point rate

3
TraesCS3B02G404600 and TraesCS3B02G404700 were differentially expressed between Linmai 2 and Zhong 892 (Fig. 2C). All three differentially expressed genes were downregulated in Linmai 2 compared to Zhong 892 (Fig. 2C). Thus, each of TraesCS3B02G404300, TraesCS3B02G404600 and TraesCS3B02G404700 could be the causal gene of QBp. caas-3BL based on sequence polymorphism and differential expression.

Development and validation of a KASP marker for QBp.caas-3BL
TraesCS3B02G404700 was identified as a candidate gene for QBp.caas-3BL, with a SNP in its ORF between the parents. To develop a breeding-applicable marker for QBp. caas-3BL, we converted the SNP into a cost-effective,  Table S3) and validated it in a panel of 166 wheat cultivars from the Huang-Huai Wheat Region, the largest wheat area in China. Association analysis indicated that sequence variation in TraesCS3B02G404700 was significantly associated with black point response (Table 1; Table S5).

QBp.caas-3BL has potential to improve black point resistance in wheat breeding
In the present study, we confirmed the effect of QBp.caas-3BL in additional two environments, in agreement with Liu et al. (2016). QBp.caas-3BL was further mapped into a physical interval of approximately 1.7 Mb from which candidate genes were also identified. These results provide important reference information for map-based cloning and marker-assisted selection of QBp.caas-3BL. Based on a polymorphic site in TraesCS3B02G404700, a candidate gene for QBp.caas-3BL, we developed a cost-effective and high-throughput KASP marker. Association analysis showed that QBp.caas-3BL had a significant effect on black point resistance using the KASP marker in a diverse panel of 166 wheat cultivars, suggesting that the marker is breeding-applicable for marker-assisted selection of black point resistance. The additional field data obtained in the study confirmed that QBp.caas-3BL is a stable major QTL for black point resistance and could be useful to improve black point resistance in wheat breeding.

Functional prediction of candidate genes for QBp.caas-3BL
The cause of black point is complicated; the symptom has been attributed to fungal infection, enzymatic browning and environmental stress, such as high humidity and temperature (March et al. 2007;Fernandez and Conner 2011;Busman et al. 2012;Fernandez et al. 2014). In this study, we mapped QBp.caas-3BL to a physical interval of 1.7 Mb and determined TraesCS3B02G404300, TraesCS3B02G404600 and TraesCS3B02G404700 as candidate genes. Phylogenetic analysis showed that TraesCS3B02G404300 and TraesCS3B02G404600 are probably resulted from tandem duplication events (Fig.  S1A). Additionally, it appears that the tandem duplication events have happened in Triticum dicoccoides (also known as wild emmer), the putative progenitor of wheat subgenomes A and B, according to BLASTN against WEWSeq_v.1.0 (Genomic sequence) (http:// plants. ensem bl. org/ Triti cum_ dicoc coides/ Tools/ Blast/ Resul ts?r= 3B: 63975 2637-63975 6075; tl= 7tHVu MlVeE 1hFS0L-20388 519). TraesCS3B02G404300 and TraesCS3B02G404600 are orthologous to ABI5 in Arabidopsis and OsABI5-1 in rice (Fig. S1A). ABI5 has pleiotropic effects on plant growth and development and is also involved in response to biotic and abiotic stresses. In Arabidopsis, ABI5 (AT2G36270) is a key regulator in ABA signaling during seed maturation and germination and modulates late embryogenesis abundant genes during both developmental stages (Finkelstein and Lynch 2000;Ibarra et al. 2016). Bi et al. (2017) showed that AtABI5 played a role in regulating the homeostasis of reactive oxygen species (ROS) by activating CATALASE1 transcription during seed germination. OsABI5 (Os01g0859300) was reported to regulate fertility and stress tolerance in rice (Zou et al. 2008). TaABF1-3A (TraesCS3A02G371800), an ortholog of TraesCS3B02G404300, was shown to participate in grain development by physically interacting with PKABA1, a signaling component in the ABA suppression of GAinduced gene expression in cereal grains ( Fig. S1; Johnson et al. 2008). Harris et al. (2013) further validated that TaABF1 functioned as an integrator of ABA and GA signaling in aleurone cells of grains. Recently, overexpression of TaABI5 (99.4% similarity with TraesCS3D02G364900 in IWGSC RefSeq v1.1) in Arabidopsis transformants exhibited high sensitivity to ABA and repressed germination (Utsugi et al. 2020). Hence, TraesCS3B02G404300 and TraesCS3B02G404600, homologs of ABI5, are probably involved in seed development by modulating ABA and GA signaling.
TraesCS3B02G404700, the third candidate gene of QBp.caas-3BL, is orthologous to OsPP16 in rice, whereas an ortholog is not detected in Arabidopsis based on phylogenetic analyses (Fig. S2). OsPP16 belongs to a dual specificity phosphatase (DUSP) clade (Singh et al. 2010). As a member of the tyrosine phosphatase superfamily, dual specificity phosphatase (DUSP) can dephosphorylate tyrosine, serine and threonine residues and is involved in many basic physiological activities such as plant growth, development and abiotic/biotic stress response (Jiang et al. 2018). OsPFA-DSP1, an atypical dual-specific phosphatase (PFA-DSP1) subfamily member in rice, may act as a negative regulator in drought stress responses (Liu et al. 2012). DUSP IBR5 in Arabidopsis is involved in regulation of R protein CHS3-mediated temperaturedependent responses (Liu et al. 2015). Thus, it appears that TraesCS3B02G404700 is involved in response to abiotic/ biotic stress, a postulated cause of black point. In summary, the functional prediction of candidate genes in QBp.caas-3BL lays a strong foundation for identification of its causal gene and ascertainment of the underlying mechanism. Transgenic experiments are ongoing to validate the functions of the candidate genes.