Fine Mapping and Distribution Analysis of Hybrid Necrosis Genes Ne1 and Ne2 in Wheat in China

doi:10.21203/rs.3.rs-896913/v1

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Fine Mapping and Distribution Analysis of Hybrid Necrosis Genes Ne1 and Ne2 in Wheat in China

https://doi.org/10.21203/rs.3.rs-896913/v1

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published 27 Jan, 2022

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Hybrid necrosis of wheat is caused by two dominant complementary genes Ne1 and Ne2 present in normal phenotype parents and is regarded as a barrier to gene flow between crop species. However, the necrosis alleles still occur at high frequency in modern wheat varieties. In this study, we constructed two high-density genetic maps of Ne1 and Ne2 in winter wheat. In these cultivars, Ne1 was found to be located in a span interval of 0.50 centimorgan (cM) on chromosome 5BL delimited by markers Nwu_5B_4137 and Nwu_5B_5114, while Ne2 co-segregated with markers Lseq102 and TC67744 on 2BS. Statistical analysis confirmed that the dosage effect of Ne alleles also existed in moderate and severe hybrid necrosis systems, and the symptoms of necrosis can also be affected by the genetic background. Furthermore, we clarified the discrete distribution and proportion of the Ne1 and Ne2 in China’s major wheat regions, and concluded that introduced modern cultivars directly affect the frequencies of necrosis genes in modern Chinese cultivars (lines), especially that of Ne2. Taking investigations in spring wheat together, we proposed that hybrid necrosis alleles could positively affect breeding owing to their linked excellent genes. Additionally, based on the pedigree, we speculated that the Ne1 and Ne2 in winter wheat may directly originate from wild emmer and introduced cultivars or hexaploid triticale, respectively.

Molecular Genetics

Medical Genetics

Hybrid necrosis

phenotype parents

crop species

pedigree

Ne1

Ne2

Flanking markers useful for identifying hybrid necrosis alleles were identified by fine mapping Ne1 and Ne2 and the distribution of the two necrosis genes was investigated in Chinese elite wheat varieties.

The hybrid offspring of some normal parents commonly exhibit a phenotype of gradually premature senescence or death. This phenomenon is known as hybrid necrosis and coincides with the Bateson–Dobzhansky–Muller (BDM) model of postzygotic hybrid incompatibility (Orr 1996). To date, several hybrid necrosis systems classified according to the extent of necrosis have been identified in plants (Bomblies and Weigel 2007). The severest type of hybrid necrosis results in death at the seedling stage resembling a form of reproductive isolation, indicating a vital role in speciation in nature (Bomblies and Weigel 2007; Chen et al. 2014; Hermsen 1963a). In terms of breeding practice, hybrid necrosis does limit the use of certain combinations of parents from diverse germplasm pools; therefore, this postzygotic incompatability was considered to be a barrier to crop improvement through combining desirable traits from different varieties or transferring genes from related species to commercial cultivars (Bizimungu et al. 1998; Hermsen 1963a). In bread wheat (Triticum aestivum L.), the inheritance of hybrid necrosis is caused by the interaction of two dominant complementary genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively (Nishikawa et al. 1974). The necrosis can start at any point during development from the 1–2-leaf stage onwards depending on the nature of crosses (Hermsen 1963a). When leaf tissues reach a certain physiologic maturity, the necrosis program is activated, then leaves begin gradual yellowing, wilting and necrosis from the tip progressively to the base of the lowest leaf to the flag leaf (Caldwell and Compton 1943). Finally, hybrid necrosis result in dwarfism of wheat plants, reduced growth rate, poor fertility/infertility or even death before the heading period (Caldwell and Compton 1943). Intriguingly, it was proposed that hybrid necrosis could be caused by autoimmunity as a result of a hypersensitive response (HR) associated with factors such as superoxide production or dysregulated signal transduction (Chu et al. 2006; Kandel 2016; Khanna-Chopra et al. 1998; Zhang et al. 2014; Zhou et al. 2015). This indicated that necrosis might be a pleiotropic effect of the evolution of genes involved in responses to pathogens (Bomblies and Weigel 2007). Hence, understanding the molecular basis and mechanisms of hybrid necrosis will improve our understanding of hybrid breeding and plant pathology.

The first genetic map of Ne1 and Ne2 was generated by Chu (2006) in spring wheat using simple sequence repeat (SSR) markers to screen two backcross populations consisting of 100 and 94 individuals, respectively. The genetic distances from Ne1 and Ne2 to their nearest SSR markers, Xbarc74 and Xbarc55, were 2.0 centimorgan (cM) and 3.2 cM, respectively (Chu et al. 2006). Recently, two high-density maps of Ne1 have been published (Li et al. 2021, Si et al. 2021a), in which Ne1 was delimited into an approximate 4 Mb interval; however, high-throughput molecular markers for screening the necrosis gene have not yet been developed. Although Ne2 has been cloned (Si et al.2021b, Hewitt et al. 2021; Yan et al. 2021), the mechanism of hybrid necrosis is still unclear and the application of Ne2 gene-specific selective markers in breeding is limited. Therefore, to develop efficient molecular markers and clone these two genes will contribute to optimizing the breeding program and understanding the mechanism of hybrid necrosis.

Surveys have shown that Ne1 and Ne2 are widely distributed among different wheat species, subspecies and cultivars throughout the world (Hermsen 1963b; Oetmann and Zeller 1989; Pukhalskiy et al. 2008a, 2018; Pukhalskiy et al. 2000; Zeven 1965). The distribution of Ne1 and Ne2 was established according to the global lists of Ne carriers published from 1963 to 1981(Hermsen 1963b; Zeven 1965, 1967, 1968, 1969, 1971, 1973, 1976, 1981). Although these surveys involved tests of 5,541 wheat cultivars (lines) worldwide, only 154 Chinese wheat cultivars were included. As a result of international cooperation, the frequencies of Ne1 and Ne2 have changed in wheat populations in different regions, although the differences in their distribution in different countries remain (Bomblies and Weigel 2007; Pukhalskiy et al. 1998, 2000, 2008b; Vikas et al. 2013). China is the top wheat-producing country worldwide; therefore, systematic analysis of the distribution and proportion of necrosis genes in wheat cultivars is important to elucidate the mechanisms by which these Ne1 and Ne2 affect wheat plant breeding and production in China.

Based on the distinct variation of necrosis in different F_l-generations (Ne1ne1Ne2ne2), Hermsen distinguished hybrid necrosis into nine grades (0–8) and three levels (grades 6–8 indicate severe necrosis, 3–6 moderate necrosis and 0–3 weak necrosis). These variations in wheat were found to be determined by the occurrence of three Ne1 alleles(Ne1w, Ne1m and Ne1s) and five Ne2 alleles (Ne2w, Ne2wm, Ne2m, Ne2ms and Ne2s) (w = weak, m = moderate, s = strong, wm and ms are inter-mediate strengths) (Hermsen 1963a). Moreover, he demonstrated that the degree in weak necrosis system is affected by the number of dominant Ne alleles (gene dosage) (Hermsen 1963a). Recently, by genotyping and phenotypic identification of the F₂ population of Pan555/Zheng891, Li confirmed that incomplete dominance at the two Ne loci controlled the timing and severity of necrosis in moderate and severe hybrid necrosis systems (Li et al. 2021). However, the dose-effect of hybrid necrosis genes in hybrids needs to be further demonstrated.

During wheat breeding to improve the resistance of high-yield cultivars using a PmAS846 donor (carried by N9134) (Xue et al. 2012), we observed several necrotic F₁ combinations by crossing normal green wheat lines. Considering the importance of wheat breeding and the gap in our knowledge of the mechanism of hybrid necrosis in wheat, we further investigated the hybrid necrosis locus in winter wheat line N9134 and compared it with that in spring wheat. In this study, we revealed the dosage effect of Ne1 and Ne2 on the intensity of necrosis in the moderate and severe necrosis system. We then constructed two detailed genetic maps of Ne1 and Ne2 in winter wheat mainly using kompetitive allele-specific PCR (KASP) markers and allele-specific PCR (AS-PCR) markers. Furthermore, we clarified the discrete distribution, proportion and genotype frequencies of the two Ne genes in the major wheat-producing areas in China. Finally, we traced the origin of Ne1 and Ne2 in the two backcross mapping populations analyzed in this study.

Plant materials

The common wheat line N9134 was selected from the hybrid progeny of 5B nullisomic (5BN) of Abbondanza/wild emmer (WE) accession AS846 (Triticum dicoccoides) (Fig. S1). N9134 and N0439 are both excellent winter wheat lines developed at Northwest A&F University. Zhoumai 22 (ZH22) and Xinong 509 (XN509) are both main commercial cultivars in the Yellow and Huai River Valleys winter wheat cultivation region of China. The F₁ generations of reciprocal crosses (e.g., N9134/ZH22 and ZH22/N9134, etc.), the corresponding F₂ populations and BC₁F₁ populations were used to find, verify, and investigate the hybrid necrosis in our wheat cultivars (Table S1). The two BC₁F₁ populations were also employed for fine mapping of Ne1 and Ne2. Specifically, the BC₁F₁ population, derived from the backcrosses of ZH22 (Ne2 carrier)//[(N9134/ZH22) or (ZH22/N9134)], was used to map the Ne1 allele in necrotic (Ne1ne1Ne2_) and normal (ne1ne1Ne2_) plants. The other BC₁F₁ population, from N9134 (Ne1 carrier) was used to map the Ne2 allele.

The two cultivars (lines), an Australian spring wheat cultivar Spica carrying Ne1s (Zhang et al. 2016), the CIMMYT synthetic hexaploid wheat (SHW) lines TA4152-60 carrying Ne1 (Chu et al. 2006), were used for allelism tests with Ne1 in N9134 (hereafter, Ne1 in N9134 is designated Ne1-nw for convenience)..

The seeds of other cultivars (lines) used for genotyping the two necrosis genes (Table S2) were obtained from the Chromosome Engineering Laboratory, Northwest A&F University or the “Precise identification and innovative utilization of wheat germplasm resources” germplasm pool (2016YFD0100102-6).

Allelism tests

To test the relationship between Ne1 in N9134, Spica and TA4152-60, N9134 was first crossed with these two Ne1-allele carriers to obtain the F₁ gametes, which were further crossed with ZH22 to form two F₁ test cross (TF₁) populations (i.e., ZH22//(N9134/Spica or Spica/N9134) and ZH22//(N9134/TA4152-60 or TA4152-60/N9134). Each TF₁ population was consisted of more than 140 progeny plants.

Investigation of the plant phenotypes and statistical analysis

All crosses, backcrosses and self-crosses were bagged. Seeds were planted at a density of one spike per row in the experimental field of Northwest A&F University, Yangling, Shaanxi, China. In previous years, the seeds were generally planted at the beginning of October, and each was then numbered in early December when the plants reached approximately 15 cm in height. The first investigation of the phenotype at the seedling stage was conducted in the early stage of winter tillering. The second investigation was conducted around the vernal equinox in following year when wheat plants were at the early jointing stage. The third investigation was carried out when plants were at the heading stage in mid-April. The investigations were conducted by direct observation of the obviously contrasting phenotypes of the necrotic and normal plants, as well as comparison with the normal parents. After summarizing the survey data, Chi-squared (ꭓ²) tests were conducted to determine the fitness of segregation ratios to theoretical Mendelian ratios.

Sampling and DNA extraction

Following the second investigation, the second leaf of plants was sampled and numbered at the jointing stage. After being freeze-dried, the samples (approximately 0.5 g of each fresh leaf in a 2.0 mL centrifuge tube) were ground using a medium throughput ball mill (TL2010, DHS Life Science & Technology Co., Ltd, Beijing, China). Genomic DNA (gDNA) was extracted using the CTAB method (Saghai-Maroof et al. 1984) with some improvements to the extraction buffer due to the use of relative old leaf tissue in this study. The specific composition of the modified gDNA extraction buffer is as follows: 0.8 M NaCl, 0.14 M D-Sorbital, 0.22 M Tris (pH 8.0), 22 mM EDTA (pH 8.0), 0.8% CTAB, 0.8% Sarcosin, 3% PVP-K30 and 2% 2-mercaptoethanol, to reduce the content of secondary metabolites.

Screening and development of molecular markers

In this study, we used some previously reported flanking molecular markers of Ne1 (Chu et al. 2006) and Ne2 (Lr13, LrLC10) (Chu et al. 2006; Qiu et al. 2020; Zhang et al. 2016). The other SSR markers were obtained from the GrainGenes Database (https://wheat.pw.usda.gov/GG3/). Specific-Locus Amplified Fragment Sequencing (SLAF-Seq) (Sun et al. 2013) was applied for typing and screening of single nucleotide polymorphism (SNP) sites between the three pools (Table S3). A total of 96,862 polymorphic SLAF tags were obtained, including 14,885 for 2B and 11,622 for 5B, in the SLAF-seq experiment, which was constructed by enzyme digestion of HaeIII with a sequencing depth of 14.31x. 5,796 and 5,979 SNP sites of the Ne1 and Ne2 alleles were obtained respectively according to the SLAF-Seq data of the positive (a) and negative (b) or (c) pools (Table S3). The upstream and downstream 500-bp flanking sequence of those SNP sites were obtained from the Chinese spring reference genome sequence (IWGSC RefSeq v1.0). The SNP sites were then converted into AS-PCR or KASP markers. The SLAF-Seq was conducted by Biomarker Technology Co., Ltd (Beijing, China). The primers were designed using PolyMarker (http://www.polymarker.tgac.ac.uk) and synthesized by Beijing AuGCT DNA-SYN Biotechnology Co. (Beijing, China). The markers used in this study are listed in Table S4. The SSR, AS-PCR and InDel molecular marker assays were performed according to previously reported protocols (Xue et al. 2012). KASP assays were performed according to the manufacturer’s protocol (LGC Genomics; http://www.lgcgroup.com).

The polymorphic markers were screened using bulked segregant analysis (BSA). DNA samples from the two parents, the heterozygous F₁, positive pool (20 necrotic BC₁F₁ plants) and the negative pool (20 normal BC₁F₁ plants) were screened separately for Ne1 and Ne2. The selected markers were further independently tested in a small number of individuals (23 necrotic plants and 22 normal plants) from the corresponding BC₁F₁ population; the markers that were still polymorphic were selected for finely mapping of the two necrosis genes in all the individuals of the two BC₁F₁ populations.

Construction of the genetic linkage maps

Linkage analysis was performed using QTL IciMapping V4.2 (http://www.isbreeding.net/software/) with grouping based on a threshold logarithm of the odds (LOD) score of 3.0, ordering by the algorithm of nnTwoOpt algorithm (nearest neighbor was used for tour construction, and two-opt was used for tour improvement) and rippling according to the sum of adjacent recombination frequencies (SARF) (Meng et al. 2015). Genetic distances were calculated using the Kosambi mapping function (Kosambi 1943).

Hybrid necrosis pedigree analysis and genotype detection of wheat cultivars

N9134 and ZH22 are regarded as the two fundamental carriers of Ne1 and Ne2 in winter wheat. The pedigrees of these cultivars was traced using hybrid tests to determine the presence of hybrid necrosis genes in their parents and ancestors (Fig. S1). Hybrid tests were also applied to classify the genotype of the wheat cultivars by carrying out crosses with Ne1 or Ne2 carriers (including, but not limited to N9134 and ZH22). Independent genotype detection was conducted using the developed close linkage KASP markers (Nwu_5B_4137 and Nwu_5B_5114 for the Ne1 locus; Nwu_2B_4204 and Nwu_2B_4249 for the Ne2 locus).

Fluorescence in situ hybridization (FISH)

To identify the differences in the karyotype of the cultivars carrying the Ne1 alleles, FISH was performed according to the protocol described by Tang et al. (2014) and using the oligonucleotide probes Oligo-pTa535 (Tamra-5', red) and Oligo-pSc119.2 (6-FAM-5', green), which were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd. (Shanghai, China). Fluorescent signals were scanned and photographed with an Olympus BX53 microscope equipped with a Photometrics SenSys CCD DP80 camera (Wang et al. 2016).

Genetic analysis of hybrid necrosis

Test crosses of WL711, Manitou and Pan555 (Ne2-carriers), as well as Spica and TA4152-60 (Ne1-carriers) (Table S1) showed that N9134 (or N0439) is a carrier of Ne1 allele, while ZH22 (or Z8425B) carries Ne2 allele and XN509 carries neither. Then, through systematic genetic analysis (Table S1) of winter wheat cultivars (lines) we confirmed that the hybrid necrosis in the winter wheat investigated in this study was also controlled by two complementary dominant alleles, and its genetic pattern was the same as that reported in spring wheat. The specific results are summarized as follows: The F₁-generation plants derived from crosses of N9134 (or N0439)/ZH22 (or Z8425B) developed necrosis consistently; the F₂ populations segregated at the ratio of 9:7(necrotic: normal) at the seedling stage; all of the BC₁F₁ populations showed a segregation ratio of 1:1 for necrosis compared with the normal phenotype; all plants in the F₁ generations of XN509/ZH22 and XN509/N0439 exhibited the normal phenotype; and the populations from XN509//N0439/ZH22 and XN509/N0439//ZH22 crosses showed the segregation ratios of 1:3 and 1:1, respectively, for necrosis compared with the normal phenotype. The hybrid necrosis traits of plants at different growth stages and cultivation conditions are shown in Fig. 1.

The dosage effect still exists in the moderate and severe hybrid necrosis system.

The F₁ plants derived from N9134 and ZH22 exhibited moderate or severe necrosis (Fig. 4c) according to the necrosis grade criteria defined by Hermsen (1963a). Through genetic analysis, we found there was one line had no living plants and only three lines exhibited all plant necrosis among the 140 F_2:3 lines derived from randomly selected necrotic F₂ plants (Table 1, Table S5). This means that there were no more than four double dominant homozygous lines. Even the ratio of double dominant homozygous lines to heterozygous lines was 4:136, it was not fit to, 1:8 (p = 7.83E^− 04), the theoretical Mendelian ratios without dosage effect. Additionally, based on this observation combined with the theoretical (F₂ = 9:7, F₃ = 25:11) and practical separation (F₂ = 8:7, F₃ = 16:11) ratios of the F₂ and F₃ populations derived from necrotic plants(Table 1, Table S1), we concluded most plants with genotype Ne1Ne1Ne2Ne2 die before heading stage and are unable to produce offspring. That is, regardless of the necrosis grades, the Ne1ne1Ne2ne2-plants (double heterozygous, dosage 2) should be the weakest generally and also coincide with the corresponding F₁-plants in terms of the necrotic intensity, while the plants with genotype Ne1Ne1Ne2Ne2 (double dominant homozygous, dosage 4) should always have the strongest degree of necrosis, and most will die before heading stage in the moderate and severe hybrid necrosis system.

Table 1

Genetic analysis of dosage effect associated with moderate and severe necrosis.
Years	Female Parent	Male Parent	Genetic population	Necrotic plants	Normal plants	Seeds	Investigation condition	Theoretical Mendelian ratios (Necrotic: Normal)	p value	Theoretical Mendelian ratios with dosage effect (Necrotic: Normal)	p value
2017-18	N0439	ZH22	F₂	110	71	264	Seedling, Field	9:7	0.220	8:7	0.045 ^*
2012-13	ZH22	N0439	F₂	137	132	380	Jointing, Field	9:7	0.079	8:7	0.429
2015-16	ZH22	N0439	F₂	128	132	420	Heading, Field	9:7	0.023 ^*	8:7	0.185
2013-14	ZH22	N0439	F₃	241	172	800 ^a	Heading, Field	25:11	9.93E^{− 07 **}	16:11	0.708
2018-19 ^b	N9134 (ZH22)	ZH22 (N9134)	F₃ (F_2:3)	986	717	2637	Heading, Field	25:11	4.44E^{− 25 **}	16:11	0.253
2018-19 ^b	N9134 (ZH22)	ZH22 (N9134)	F_2:3	3 ^c	136 ^c	140 ^c	Heading, Field	1:8	7.83E^{− 04 **}	0:8	——
Notes: a. The seeds were randomly selected from the mixed seeds of the F₂ necrotic plants. b. The same population, investigated as individual plants and lines. The details of the F_2:3 population are shown in Table S5. c. The number of the F_2:3 lines having necrotic plants only; The number of F_2:3 lines containing both necrotic and normal plants; The number of spikes selected from necrotic F₂ plants. P -values obtained by chi-squared analysis of the ratios of the genetic population and theoretical Mendelian ratios. ^P < 0.05; ^*P < 0.001.

High-density genetic maps of the Ne1 and Ne2 in winter wheat.

Based on the linked markers of Ne1 (Chu et al. 2006) and Ne2 (Lr13 and LrLC10) (Chu et al. 2006; Qiu et al. 2020; Zhang et al. 2016) reported in spring wheat, we constructed two genetic maps of these genes in winter wheat using 269 and 264 BC₁F₁ plants, respectively (Fig. S2). Two high-density genetic linkage maps of these genes (Fig. 2) were then constructed mainly using the developed KASP markers and AS-PCR markers in the two BC₁F₁ populations consisting of 1,006 and 1,143 plants, respectively. The detailed genetic map of the Ne2 locus contained 25 molecular markers (Fig. 2a, Fig. 2b). Both the InDel marker Lseq102 (co-segregated with LrLC10) and SSR marker TC67744 co-segregated with Ne2. These markers were located at approximately 156.59 and 157.76 million bases (Mb), respectively, on chromosome arm 2BS in Chinese spring RefSeq v1.0. Furthermore, Ne2 was located between the two flanking tightly linked InDel markers, Lseq54 and Lseq22, with a genetic interval of 0.18 cM and a physical distance of about 4.45 Mb. The SNP markers Nwu_5B_4137 and Nwu_5B_5114 were identified as the two closest linked markers on either side of the Ne1 locus, with the genetic interval of 0.50 cM (0.20 and 0.30 cM). Both markers were found to be located separately at approximately 383.40 Mb and 388.01 Mb on chromosome arm 5BL in Chinese spring RefSeq v1.0, with the physical interval 4.61 Mb (Fig. 2c, Fig. 2d).

Distribution, proportion and genotype frequencies of Ne1 and Ne2 are discrete in China’s wheat regions.

Based on the planting area and yield of China’s 10 agro-ecological production zones (CHINA STATISTICAL YEARBOOK 2019, http://www.stats.gov.cn/tjsj/ndsj/2019/indexch.htm), we chose wheat cultivars (lines) for investigation according to the proportion grown in each area from the “Precise identification and innovative utilization of wheat germplasm resources” program germplasm nursery. The selected materials (consisting of 1,178 modern Chinese cultivars (MCC), 65 Chinese landraces (CL) and 121 introduced modern cultivars (IMC) ) were genotyped using molecular marker and/or hybrid test. Among them, 99 were detected by the both two methods, and 98 out of the 99 had the consistent genotyping for Ne1 or Ne2 (Table S2). In accordance with this, the two groups of genotype frequencies obtained separately using the two methods were also showed a highly positive correlation (r = 0.919, Fig. 3a). After de-redundancy, 26.2% of the cultivars (lines) were genotyped as Ne1Ne1ne2ne2, while 33.2% were genotyped as ne1ne1Ne2Ne2 (Fig. 3b). These results in Fig. 3c demonstrated discrete differences in the distribution and proportion of Ne genes from different wheat region, with highly variable correlation coefficients among the regions (Fig. 3d). Typically, Ne1Ne1ne2ne2 was significantly dominant in the region IV, whereas ne1ne1Ne2Ne2 was significantly dominant in the IMC population. Cultivars (lines) in the region III had the highest frequency of ne1ne1ne2ne2 (Fig. 3e).

Similarly, according to the CL, IMC and MCC classification (Fig. 3f), we found that the CL population had the highest frequency of both the ne1ne1ne2ne2 and Ne1Ne1ne2ne2 genotypes, while the IMC population showed the relatively lowest frequency of ne1ne1ne2ne2, but with the highest frequency of ne1ne1Ne2Ne2. Intriguingly, the Ne2 gene frequency in the MCC population was 2.3 times higher than that in the CL population due to the introduction of the IMC into wheat breeding program of China. Moreover, the proportions of the three genotypes distributed in the MCC population were highly correlated with the average of those in the CL and the IMC (r = 0.971; Fig. 3g). These findings indicated that the genotype frequencies of Ne genes in the MCC are formed by the interaction of the CL (contributing Ne1 allele) and the IMC (contributing Ne2 allele).

Ne1-nw is inherited from WE As846, while Ne2 in ZH22 is inherited from the IMC or hexaploid triticale

To clarify the origins of Ne1-nw and Ne2 in ZH22, we traced the pedigree of these genes separately. The pedigree of N9134 indicated that Ne1 is inherited from WE As846 (Fig. S1a). We found that chromosome 5B of N9134 can only be inherited from WE As846, which was consistent with a previous study of PmAS846 (Xue et al. 2012). In addition, when ZH22 was crossed with WE As846, the F₁ plants showed necrosis similar to the F₁-progeny of ZH22/N9134, while the offspring of ZH22/Abbondanza 5BN were normal. This result also confirmed that Ne1-nw is inherited from WE As846. However, based on these results, we could only infer that the Ne2 in ZH22 is inherited from one of the five ancestral parents, four introduced modern cultivars or one hexaploid triticale. The F₁-plants obtained by crossing N9134 and Z8425B showed hybrid necrosis, while those of the other three crosses between N9134 and Zhoumai 12, Yumai 49, and Zhoumai 9 did not; therefore, we proposed that Ne2 in ZH22 was inherited from Z8425B. However, since Z8425B is derived from five ancestral cultivars (2 Italian cultivars (lines) named St2422/464 (Zhengyin 4) and St1472/506 (Zhengyin 1), 1 Russian named Прелгорная 2 (Erythrospermum-315H160/Wumang 1), 1 Mexican cultivar named Nainari60, and a hexaploid triticale line Guangmai 74) (Fig. S1b), the exact donor of Ne2 remains to be verified.

Genetic background causes different hybrid necrosis phenotypes

In our study, we observed differences in necrotic phenotypes between the two BC₁F₁ populations only with different female parents (Fig. 4). Specifically, the necrosis processes in the BC₁F₁ plants with female N9134 parents involved yellowing in the leaves became yellow from the tip to base, although the whole leaf remained fresh and moist for a short period, followed by gradually drying from the tip. These observations indicated that this type of necrosis occurs independently through two independent processes that result in yellowing and dryness (Fig. 4a). However, the processes in the BC₁F₁ plants from backcross of ZH22 involved gradually yellowing and drying of the leaves almost simultaneously from the tip to base. These observations indicated that this type of necrosis occurs through only one process (Fig. 4b). This phenomenon was also being observed in F₁ offspring of different female parents (Fig. 4c). Since the dosage and environmental condition were identical in these two models, we proposed that N9134 and ZH22 confer differential sensitivities to the activated senescence process, thus representing an example of the effect of genetic background on the manifestation of hybrid necrosis.

Ne1-nw is implicated as a novel hybrid necrosis allele

To date, three Ne1 alleles (Ne1w, Ne1m and Ne1s) have been reported in wheat (Hermsen 1963a). The existence of other alleles is suggested by the differences in the multiple alleles of Ne1, not only at the phenotypic level identified in allelism tests, but also the linkage molecular markers (Chu et al. 2006; Huang et al. 2020; Qiu et al. 2020; Zhang et al. 2016). The allelism tests (Table S6) performed in this study indicate that the loci of Ne1s in Spica and Ne1 in TA4152-60 are distinct from Ne1-nw. Additionally, karyotype analysis revealed that the genetic backgrounds of these genes are very different (Fig. S3). Since the Spica and TA4152-60 are spring wheat cultivars (lines), it can be inferred that Ne1-nw has a different origin to the other two Ne1 alleles.

Due to the difference in the Ne1 allele frequencies between tetraploid (emmer, durum and timopheevi) and the hexaploid wheat (Hermsen 1963a; Mori and Tsunewaki 1992; Tsunewaki 1970; Zeven 1969), it is generally accepted that the multiple Ne1 alleles differentiated genetically before domestication. In this study, we demonstrated that the Ne1-nw was derived directly from WE (tetraploid wheat). Comparison of the Ne1 locus interval in Chinese Spring (IWGSC RefSeq v1.0) with that in WE (Zavitan WEWSeq v1.0), revealed a fragment (residing an initiation factor 4F subunit-encoding gene, an alpha/beta-hydrolases superfamily protein-encoding gene and three HtrA genes) as a tandem duplication in Chinese Spring. It is not surprising that the structure and function of these repeat genes changed over the course of evolution. We further speculate that the tandem repeat genes in the Ne1 loci perform parallel or redundant functions. Therefore, this indicates that these Ne1 alleles are different, thus implicating Ne1-nw derived from WE as a novel hybrid necrosis gene.

Ne1 may be related to serine protease HtrA

We mapped Ne1 to a physical interval from 383.40 Mb to 388.01 Mb on chromosome 5B of Chinese spring RefSeq v1.0, which was consistent with the interval mapped by Li and Si (Li et al. 2021; Si et al. 2021a). The Ne1 intersection region of the three genetic maps contains 14 candidate genes (2 encoding initiation factor 4F subunit, 2 alpha/beta-hydrolases superfamily protein, 1 late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein and 9 serine protease HtrA). The Ne1 allele in ZN17 was associated with a 2.89 Mb deletion on chromosome arm 5BL containing four high-confidence genes (1 alpha/beta-hydrolases superfamily protein-encoding gene and 3 HtrA genes) (Si et al. 2021a). Additionally, all the three HtrA genes in the deletion were significantly differentially expressed between the positive and negative pools according to our BSR-seq data (unpublished). HtrA proteases share common mechanisms of activation with classic serine proteases such as trypsin, chymotrypsin and elastase (Clausen et al. 2011). It was reported that HtrA proteins participate in defense against stresses causing aberrations in protein structure, and disturbances in their function may induce carcinogenesis or arthritic and neurodegenerative disorders (Zurawa et al. 2007). Chen (2014) demonstrated that the interaction of Hwi1 (comprises 2 LRR-RLK genes) and Hwi2 (encodes a putative subtilisin-like protease with serine protease activity) can activate the autoimmune response in the basal nodes of hybrids in rice. Recently, Ne2 has been cloned and confirmed to be a typical CC-NBS-LRR-type R gene (Si et al.2021b, Hewitt et al. 2021; Yan et al. 2021). Therefore, we speculate that Ne1 is a serine protease and the enzyme or its substrate is specifically recognized by Ne2, leading to the constitutive activation of the disease resistance response. This hypothesis remains to be tested in further investigation of isolated Ne1.

The IMC should directly affect the frequencies of necrosis genes in MCC, especially Ne2.

In the nine reports from 1963 to 1981, 1,467 of the 5,541 cultivars (26.5%) reported globally carried Ne1 and 1,189 (21.5%) were Ne2 carriers all over the world, while 60 carried Ne1 (39.0%) and 15 carried Ne2 (9.7%) among the 154 Chinese wheat cultivars (lines) (Hermsen 1963b; Zeven 1965, 1967, 1968, 1969, 1971, 1973, 1976, 1981). In contrast, among the 1,178 MCC used in this study, 311 cultivars (26.4%) carried the Ne1Ne1ne2ne2 genotype and 378 cultivars (32.1%) carried the ne1ne1Ne2Ne2 genotype (Table S2, Fig. 3f). Strikingly, in comparison with these previous reports, the frequency of the Ne1 carriers in China showed a decrease of 12.6% (from 39.0–26.4%), while the frequency of Ne2 carriers rose considerably from 9.7–32.1%. These trends are consistent with those of previous reports (Bomblies and Weigel 2007). These findings imply that these two Ne-genes were unconsciously selected by breeders, and Ne2 has greater selective advantages than Ne1, probably due to the leaf rust resistance conferred by Ne2 (Lr13) (Si et al.2021b, Hewitt et al. 2021; Yan et al. 2021) or other tightly linked loci conferring advantageous traits. This inference is also be supported by the modern cultivars derived from the backbone parent, Z8425B, in wheat breeding of China (Fig. S1b). Moreover, considering the frequency variation between the CL, IMC and MCC population (Fig. 3f), we speculate that the increase in the Ne2 necrosis allele in China is caused by the decisive effect of IMC. This indicates the important contribution of the IMC to wheat breeding in China, as well as the robust constitution of the MCC genome (Chen et al. 2019; Hao et al. 2020).

Hybrid necrosis alleles could also positively affect wheat breeding.

Hybrid necrosis is generally considered to be a barrier to gene flow at the inter- or intra-specific levels (Presgraves 2010; Rieseberg and Willis 2007; Zhou et al. 2020) and is usually regarded as a negative influence on breeding (Hermsen 1963a; Vikas et al. 2013) that must be avoided. Nevertheless, necrosis occurs only when both Ne1 and Ne2 are present in the same plant (Hermsen 1963a), which alleviates the necessity for elimination of both alleles simultaneously. In fact, both the ‘negative effectors’ Ne1 and Ne2 are linked with advantageous genes (Bomblies and Weigel 2007; Xue et al. 2012; Zeven 1981; Zhang et al. 2016); that is, both could positively affect breeding when they occur independently in an individual. Therefore, if breeders reserve F₁ plants showing hybrid necrosis, the high-quality plants might be separated in F₂ generation, increasing the chance of obtaining progeny with superior comprehensive traits. Taken together, the information presented here represents a possible foundation from which the traditional cognition of hybrid necrosis can be adjusted to allow wheat breeders to avoid missing potential elite offspring of necrotic plants. In addition, the Ne1 and Ne2 are still widely distributed all over the world instead of being eliminated (Bomblies and Weigel 2007; Vikas et al. 2013). This fact could also support this view, meanwhile it also hints that the diversity of the excellent germplasm resources should be not sufficient enough for wheat breeding (Hao et al. 2020; Zhou et al. 2020).

Author contribution statement:

W.J., H.Z., M.Z. and S.L. designed the research. M.Z., S.L., YZ.W. and S.W. performed the research. M.Z., S.L., C.C., C.W. and YJ.W. contributed to the development and investigation of materials. M.Z., S.L. and YZ.W. contributed by collecting the samples for DNA extraction. M.Z., S.L. and Y.Z.W. developed the molecular markers and constructed the genetic maps. S.W. conducted fluorescence in situ hybridization. M.Z., S.L. and H.Z. analyzed the data and contributed to writing the article.

Acknowledgments

This work was supported financially by the National Key Research and Development Program of China (grant no. 2016YFD0100302, 2016YFD0102004 and 2016YFD0100102), and by the Key Research and Development Program of Shaanxi Province (grant no. 2019ZDLNY04-06).

We would like to thank: Dr. Peng Zhang (Plant Breeding Institute, University of Sydney, Cobbitty, NSW, Australia) for providing Spica, TA4152-60 and WL711 seeds; Dr. Yonggui Xiao (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China) for providing the Manitou seeds; Prof. Xinglai Pan (Food Crop Science Department, Cotton Research Institute, Shanxi Agriculture Science Academy, Yuncheng, Shanxi, China) for providing the Pan555 seeds; and thank Dr. Xiaojie Chen (Institute of isotope research, Henan Academy of Sciences, Zhengzhou, Henan, China) for providing Yutong 68-2, Yutong 194 and Yutong 198 seeds. We would also like to thank Prof. Steven S. Xu, Dr. Andrew Green, Dr. Ahmed Amri, Dr. Mergoum Mohamed, Dr. Horsley Richard, Dr. Efren Rodriguez Carranza, Dr. Nicole Boyer and others not mentioned here for their kind assistance in obtaining the wheat seeds carrying the necrosis genes.

In addition, we are grateful to Cong Li, Bo Liu, Jingxuan Chen and others for their help in this investigation and the preliminary experiment preparation. We are also grateful to Dr. Miaomiao Nie and Dr. Xudan Kou for their advices on and critical review of this manuscript.

Conflict of interest:

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Fig.S1.tif
Fig. S1 The pedigrees of N9134 (A) and Zhoumai 22 (B).
Fig.S2.tif
Fig. S2 The genetic linkage maps of Ne1 and Ne2 in winter wheat using 269 and 264 BC1F1 plants.
Fig.S3.tif
Fig. S3 Fluorescence in situ hybridization (FISH) analysis using the oligonucleotide probes Oligo-pTa535 (red) and Oligo-pSc119.2 (green).
TableS1.xlsx
Table S1. Genetic analysis and confirmation of Ne genes.
TableS2.xlsx
Table S2. Details of 1,364 cultivars (lines) used for genotyping the two hybrid necrosis genes Ne1 and Ne2.
TableS3.xlsx
Table S3. The three sample pools used in the SLAF-Seq.
TableS4.xlsx
Table S4. The markers used in this study.
TableS5.xlsx
Table S5. Details of the dosage effect in the F2:3 population.
TableS6.xlsx
Table S6. Allelism tests for distinguishing the multiple alleles of Ne1.

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Fine Mapping and Distribution Analysis of Hybrid Necrosis Genes Ne1 and Ne2 in Wheat in China

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Abstract

Figures

Key Message

Introduction

Materials And Methods

Plant materials

Allelism tests

Investigation of the plant phenotypes and statistical analysis

Sampling and DNA extraction

Screening and development of molecular markers

Construction of the genetic linkage maps

Hybrid necrosis pedigree analysis and genotype detection of wheat cultivars

Fluorescence in situ hybridization (FISH)

Results

Genetic analysis of hybrid necrosis

The dosage effect still exists in the moderate and severe hybrid necrosis system.

High-density genetic maps of the Ne1 and Ne2 in winter wheat.

Distribution, proportion and genotype frequencies of Ne1 and Ne2 are discrete in China’s wheat regions.

Ne1-nw is inherited from WE As846, while Ne2 in ZH22 is inherited from the IMC or hexaploid triticale

Genetic background causes different hybrid necrosis phenotypes

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

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