CN112273221A

CN112273221A - Breeding method of high-hardness wheat

Info

Publication number: CN112273221A
Application number: CN202011226440.0A
Authority: CN
Inventors: 亓增军; 刘鑫; 张慧婷
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-01-29
Anticipated expiration: 2040-11-05
Also published as: CN112273221B

Abstract

The patent discloses a breeding method for breeding high-hardness wheat. The wheat bred by the method contains a wheat-rye translocation chromosome T5DL & 5DS-2RS, and the translocation chromosome is lack of hardness genes Pina and Pinb carried by 5DS and a soft protein gene Gsp-1, so that the grain hardness is obviously improved, the protein content is high, the method is a novel method for breeding common wheat similar to durum wheat, and the disclosed 1 molecular marker combination can accurately distinguish T5DL-5DS-2RS homozygote, heterozygote and non-translocation single plant at the same time through one-time amplification, is beneficial to marker-assisted selection and utilization of T5DL & 5DS-2RS in high-hardness wheat breeding, provides a novel method for wheat quality improvement, and belongs to the technical field of crop genetic breeding and agricultural biology.

Description

Breeding method of high-hardness wheat

One, the technical field

The patent discloses a breeding method of high-hardness wheat, and belongs to the technical field of crop genetic breeding and agricultural biology. The application identifies and discovers that the hardness (hardness index 83.28 +/-1.63) of grains of a wheat-rye translocation line T5 DL.5 DS-2RS is obviously higher than that of Yangmai 158(68.32 +/-2.45) and Nicotiana 19(71.25 +/-0.82) due to the lack of hardness genes Pina and Pinb and a soft protein gene Gsp-1 carried by 5DS, the content of the grains of the wheat-rye translocation line T5 DL.5 DS-2RS is increased, and 2 hybrids F are obtained by hybridizing the T5 DL.5 DS-2RS with the Yangmai 158 and the Nicotiana 19₃The group, wherein the hardness indexes of the T5 DL.5 DS-2RS homozygote sub-group are respectively and remarkably improved by 11.70 and 16.34 to 79.60 +/-7.27 and 85.27 +/-4.11 compared with the hardness indexes of the sub-group not containing the translocation, and the disclosed 1 molecular marker combination can simultaneously and accurately distinguish T5 DL.5 DS-2RS homozygote, heterozygote and translocation-free single strain through one-time amplification, and is favorable forThe marker assisted selection and utilization of T5DL & 5DS-2RS in the breeding of high-hardness wheat provides a new method for improving the quality of wheat.

Second, background Art

Grain hardness is a major quality characteristic of common wheat (Triticum aestivum L). According to different hardness, common wheat can be divided into hard wheat, mixed wheat and soft wheat, and hard wheat has super-hard grains due to the lack of D genome, and belongs to a single type. Grain hardness is an important factor affecting wheat milling and baking quality. Hard wheat has high flour yield and high protein content, and is suitable for making bread and high-quality noodle foods, while soft wheat is suitable for making biscuit foods (see the references: Henry R, Furtado A, Rangan P. wheat cut transformed dough products controlling key tracks for human reference and crop adaptation [ J ]. Current Opinion in Plant Biology, 2018, 45: 231-.

Hexaploid wheat grain hardness is mainly controlled by two genes, Pina and Pinb, on 5DS (see references: Giroux M, Morris c. wheat grain hardless stresses from high after consistent stresses in the conventional compositions puroindoline a and b [ J ] Proceedings of the National Academy of Sciences of the United States of America, 1998, 95 (11): 6262-. The Pin gene is positioned at the end of 5DS, belongs to a single copy gene in common wheat and is a main effect gene for controlling the hardness of grains, and different variation types can be generated by gene mutation to change the hardness of the grains. Durum wheat is a conical wheat variety, only comprises two genomes A and B, because of the lack of the genome D, the Pina and Pinb genes are simultaneously deleted, so that grains are super-hard, the protein content and the gluten content are high, and the durum wheat is a macaroni and a high-quality noodle raw material (see references: Qixiaoyue, Jianboli, Xinlinging and the like.

The Pin gene can code a tryptophan-rich alkaline protein, puroindoline protein. The Pin gene is specifically expressed in endosperm, and the hardness of the kernel is not only related to the coding sequence, but also closely related to the expression level. Transcriptional analysis indicated that The influence of The level of Pin gene expression on The grain hardness was more significant (see references: Wang D, Zhang K, Dong L, Dong Z, Li Y, Hussain A, ZHai H. molecular genetic and genetic analysis of what milk milling and end-use traces in China: Progress and fields [ J ]. The Crop Journal, 2018, 6 (1): 68-81). The wheat with Pina (Pina-D1a) and Pinb (Pinb-D1b) alleles is wild type, and the grains are soft. In the course of ordinary wheat domestication, both Pina and Pinb may mutate, leading to stiffening of the grain (see references: Chen F, Zhang F, Morris C, He Z, Xia X, Cui D. Molecular characterization of the puroindoline a-D1b alloy and definition of stands marker in leather L. [ J ]. Journal of Cereal Science 2010, 52 (1): 80-82; Chen F, Zhang F, Xia X, Dong Z, Cui D.distribution of puroindoline alloys in broken leather of the Yellow and Huai Valley of China and Science of the scientific of the non-related pigment a leather J.J.: idea of pigment J.J.: J.S.: 29. evaluation of wheat protein J.S.: J.: 1. evaluation of wheat protein J.S. J.: 7. J.S.S. J.S. J.: 7, 51: 235-237). A number of types of variation have been found, such as Pinb-D1c, Pinb-D1D, Pinb-D1e, Pinb-D1f, etc. (see references: Morris C. puroindolines: the Molecular genetic basis of what grain hardness [ J ]. Plant Molecular Biology, 2002, 48 (5-6): 633-647), the two most widespread genotypes being Pina-D1b/Pinb-D1a and Pina-D1a/Pinb-D1 b.

The Gsp-1 gene is also a key gene for controlling wheat grain Hardness, and Giroux & Morris analyzed the Hardness gene, identified Pinb, found Pina, and excluded that Gsp-1 is a candidate gene for Hardness loci (see references: Giroux M, Morris C. wheat grain hardless stresses from high consistent statistics in the family of the branched peptides, puroindoline a and b [ J ]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95 (11): 6262-. Unlike the Pin gene, the Gsp-1 gene is present not only on 5DS but also on the 5A and 5B chromosomes of bread wheat, and several studies have shown that the absence or allelic variation of Gsp-A1 and Gsp-B1 has no significant effect on grain texture, indicating that these genes do not play a critical role in grain hardness (see references: Tranquili G, Heaton J, Chicaiza O, Dubcovsky J. Substistions and relationships of genes related to grain hardness in flour and the ir effect grain texture [ J ]. Crop Science, 2002, 42: 1812-. The GSP-1 protein is 164 amino acids in length compared to 148 residues of the PINA and PINB proteins (see references: Turner M, Mukai Y, Leroy P, Charef B, applications R, Rahman S. the Ha logic of the heat: identification of a polymeric region for a harvesting grain in cross in Genome, 1999, 42: 1242-1250), with about 60% similarity to the PIN protein. In hexaploid wheat, deletion of a large fragment of the short arm of chromosome 5 from the A and B subgenomes resulted in the loss of the Pin gene but not the Gsp-1 gene (see references: Chantret N, Salse J, Sabot F, Rahman S, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudier P, Gautier M, Cattolico L, Beckert M, Aubourg S, Weissenbach J, CaboM, Bernard M, Leroy P, Chalhoub. molecular basis of evolution expression of the Harconnected in binary and polyplewy (Triticum aegyops) [ J ]. cell 2005, 1033.1045).

The hardness range of wheat in China is large, and hard wheat, soft wheat and mixed wheat coexist. Wherein hard wheat is suitable for being planted in northern wheat areas of China and soft wheat is suitable for being planted in southern wheat areas of China (see reference documents: Zhouyanhua, Hades, Zhanyan, Wangdsen and Zhonggui English)]Chinese agricultural science, 2002 (10): 17-25). Chen et al (2006) analyzed 219 wheat local varieties and 166 cultivars in China as well as 12 foreign varieties for grain hardness and Puroindoline allelic variation, indicating that the local varieties had soft, mixed and hard genotype frequencies of 42.7%, 24.3% and 33.0%, respectively, and PINA null, Pinb-D1b and Pinb-D1p frequencies of 43.8%, 12.3% and 39.7%, respectively (see references: Chen F, He Z, Xia X, Xia L, Zhang X Y, Lillemo M, Morris C F. molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars[J]The therapeutic and Applied genetics.2006, 112 (3): 400-409). Xia et al (2005) found 79 soft wheat, 53 mixed wheat, 119 hard wheat out of 251 varieties of Chinese winter wheat, and 3 mutation types were observed in hard wheat, of which Pina-D1a/Pinb-D1b was the main type (see references: Xia L, Chen F, He Z, Chen X, Morris C F. Occurence of puroindoline allels in Chinese turbines [ J ] L]The cellular Chemistry Journal, 2005, 82: 38-43). Hardness measurements of 267 triticale varieties from CIMMYT, Russia and Ukrainian by Chen et al (2012) found that Pinb-D1b is the most widespread type, while PINA null is the predominant genotype in CIMMYT lines (see references: Chen F, Zhang F, Xia X, Dong Z, Cui D.distribution of puroindoline alloys in broken heat of the Yellow and Huai Valley of China and discovery of novel puroindoline alloys with PINA protein [ J.D.]Molecular Breeding, 2012, 29: 371-378). Thus, there is a limited global view of the allelic variation of the durum gene in common wheat, especially the lack of simultaneous deletion of Pina and Pinb types. Tranquili et al (2002) found that hardness index values of the China spring deletion line 5DS-2 and the China spring Red Egyptian5D substitution lines both lacking Pina-D1 and Pinb-D1 were significantly increased, and indicated that the China spring Red Egyptian5D substitution line can be used to cultivate common wheat like durum wheat (see references: Tranquili G, Heaton J, Chicaiza O, Dubcovsky J. Substistitutions and deletions of genes related to grains in wheat flour and the hair effect on grain texture [ J ] J].Crop Science，2002，42：1812-1817)。Morris&Beecher (2012) utilizes Chinese spring 5DS-2 and soft white spring wheat variety Alpowa to make hybridization and backcross so as to culture 5DS-2 and 5D near isogenic line, BC7F₂The segregating population demonstrated that 5DS-2 transmission was normal and consistent with an increase in kernel hardness, and that the kernel hardness index was 88.6 for the 5DS-2 type, while only 29.4 for the intact 5D type. Meanwhile, 5DS-2 type seeds are cutin, and complete 5D type seeds are powdery, which shows that 5DS top segment not only comprises seed hardness gene, but also comprises seed cutin rate control gene,however, the 5D type has a 20% higher weight and a slightly larger grain size than the 5D deletion, and the protein content is not very different (see references: Morris C, Beecher B. the difference of the meal L.) chromosome 5D short arm control end kernel vision and grain hardness [ J ] for the 5D type]The therapeutic and Applied Genetics, 2012, 125: 247-254). About 20% of the 5DS-2 deletion segment is reported (see the references: Endo T R, Gill B S. the deletion stocks of common where. J Hered, 1996, 87: 295-307), and the 5DS deletion segment is presumed to be larger to cause more gene deletion, thereby causing certain adverse effects on the development of grains or traits thereof. Genome sequencing analysis found that the grain hardness genes Pina-D1, Pinb-D1 and the soft protein gene Gsp-D1 are located in a very small segment at the top of the 5DS (see references: Gautier MF, Aleman ME, Guirao A, Marion D, journal P (1994) Triticum aestivum puroindolines, two basic cytoline-rich proteins: cDNA sequence analysis and hierarchical gene expression. plant Molecular Biology 25: 43-57; Rahman S, Jolly JC, Skerrit, Wallosock A (1994) Cloning of a 15-kDa protein software protein (GSP) and GSP a mutation of gene expression of J. G. Sa. G. J. the important gene genes are deleted, thus the loss of the soft protein gene Gsp-D917 can be reduced by the simultaneous application of the three genes Pinb-D1 and the loss of the soft protein gene expression genes III. G. Skinq. the strain of the wheat gene expression of the wheat variety Gsp-D1 can be used to simultaneously improve the quality of the wheat variety of wheat variety Pilnb-D. the wheat variety . Through many years of effort, Nanjing university of agriculture develops a wheat-rye small-fragment translocation line T5 DL.5 DS-2RS (see references: Zhuang L, Liu P, Liu Z, Chen T, Wu N, Sun L, Qi Z. multiple structural associations and physical mapping of eye chromosome 2R expressed in wheat flour J]Molecular Breeding, 2015, 35: 133-144), and molecular marker analysis shows that Pina-D1 and Pinb-D1 are deleted, while the translocated rye 2RS fragment obviously increases the content of grain protein and lysine, and the grain hardness is increased (see reference: accurate identification and effect analysis of Caocan, wheat-rye small-fragment translocation line T5 DL.5 DS-2RS [ D]Nanjing (Hayao-Kagaku)University of agriculture major academic thesis, 2018). The present application further identified this translocation breakpoint, locating the translocation breakpoint on 2RS at 2RS FL 0.6, and the breakpoint on 5D at the 5DS apical AFA family signal region, but with specific size cytology difficult to identify. Using the position of Pin gene, the gene sequence (Triticum aestivum Ensembl plants, Release 30) of about 1bp to 5Mb on Chinese spring 5D reference sequence is adjusted, and compared with Chinese spring A, B genome reference sequence (TGACCV 1, https:// heat-source-versals. inra. fr/Seq-reproducibility/assembies), 11 pairs of amplification primers are designed, wherein 8 pairs of primers have amplification on wheat (Table 1), wherein 467979 of primer amplifies three bands of 5A, 5B and 5D in Chinese spring, and only two bands of 5A and 5B are amplified in both N5DT5B and T5 DL.5 DS-2RS, and the lack of 5D band indicates that the amplification product of the primer on 5D is located in the deleted segment, and the amplification product of primer 7980 is located in the deleted segment, so that the 5D segment is located between 5Mb and 5Mb displacement segments, and the total hardness of the gene comprises about 96.387 and 96.3 Mb of the deletion gene, and the total hardness of about 96.3. Mb including the deletion of the gene segment of Pin, and the deletion of 5D, Pinb and the Soft protein Gene Gsp-1 (http:// 202.194.139.32/searchools /). The grain hardness analysis shows that the T5 DL.5 DS-2RS grain hardness is obviously higher than that of hard wheat Yangmai 158 and Ninong 19, and F obtained by hybridization of T5 DL.5 DS-2RS, Yangmai 158 and Ninong 19₃The mixed segregation population analysis shows that the hardness of homozygous translocation type seeds containing T5 DL.5 DS-2RS is obviously higher than that of non-translocation types, the agronomic traits are not obviously different, and strains with obviously improved traits are bred, the transmission rate of T5 DL.5 DS-2RS is slightly reduced through genetic analysis, but translocation chromosomes in homozygous T5 DL.5 DS-2RS are normally transmitted, so that the method can be used for breeding high-hardness wheat varieties. The patent also discloses that one molecular marker combination can simultaneously identify T5 DL.5 DS-2RS homozygote, heterozygote and translocation-free type through one-time PCR amplification, and provides a powerful tool for T5 DL.5 DS-2RS molecular marker assisted breeding.

Third, the invention

The technical problem is as follows:

the invention aims to disclose a breeding method of high-hardness wheat.

The technical scheme is as follows:

first, a wheat-rye translocation line T5 DL.5 DS-2RS (see references: Zhuang L, Liu P, Liu Z, Chen T, Wu N, Sun L, Qi Z. multiple structural associations and physical mapping of eye chromosome 2R expressed in the world wheat [ J ]. Molecular Breeding, 2015, 35: 133) created by Nanjing university of agriculture is hybridized with Nicotiana 19, Yangmai 158, wheat 9023 (publicly known, my wheat variety) and seeds of F2 and F3 populations are obtained by serial planting. At the same time, the identification and application of the oligonucleotide set for simultaneous identification of wheat and rye chromosomes [ D probe, G-Shu university, T2019. oligonucleotide set for identifying wheat and rye chromosomes [ D ] probe, G-Shu university, T2015. D ] WO 83 and S-Ha chromosome translocation on the chromosome of AFM family, S.A. G.S.A. 5, and S.A. 19. F.S.A. 5, and S.D.S.A. 5 (FIS.A. 5) were carried out by using the rye genomic probe, the oligonucleotide set #4(ONPM # 4; see references: Du P, Zhuang L, Wang Y, Wang Q, Wang D, Dawawadoudonup, Tan L, Shen J, Xu H, Zhao H, Chu C, Qi Z. development of oligonucleotides and Mux probes for obtaining and clinical diagnosis of clinical diagnosis and diagnosis of disease in vitro and diagnosis of disease, and diagnosis of disease, diagnosis and diagnosis of disease, diagnosis, however, the specific size was difficult to identify from cytological results, with the 2RS translocation segment accounting for approximately 40% of the short arm and the breakpoint at 2RS FL 0.6 (fig. 2). Meanwhile, based on The alignment results of rye whole genome sequences (Bauer E, Schmutzer T, Barilar I, Mascher M, Gundlach H, Martis 4M, Twardziok S, Hackauf B, Gordillo A, Wilde P, Schmidt M, Korzun V, Mayer K, Schmid K, Schon C, Scholz U.S. Towards a book-genome sequence for eye (Secale cereale L.) [ J ]. The Plant Journal, 2017, 89 (5): 853. invertebra. versals. inra. fr/Seq-chromosome/templates) and wheat whole genome sequences (TGACCv 1, https:// white-user-genome. versals. inra. frq-chromosomes) were co-designed and screened to explore chromosome positions in chromosome R2 (Table 2R 2) for further search.

Next, in order to determine the breakpoint position of the translocation line on the 5D chromosome, a gene sequence (Triticum aestivum Ensembll plants, Release 30) of about 1bp to 5Mb on 5D was retrieved according to the position of the Pin gene, and compared with a Chinese Spring (CS) A, B genome reference sequence (TGACCV 1, https:// heat-urgent. versals. ira. fr/Seq-reproducibility/assembies), 11 pairs of 5D specific amplification primers were designed, and the interval of the translocation breakpoint on 5DS was determined according to the amplification result marked on the translocation line (Table 1; FIG. 3). Meanwhile, specific amplification primers of hardness gene Pin and soft protein gene GSP-1 reported by the former (see references: Chen F, Zhang F, Morris C, He Z, Xia X. Cui D. Molecular characterization of the puroindoline a-D1b alloy and depth of anchors in leather L. [ J ]. Journal of Cereal Science 2010, 52 (1): 80-82; Chen F, Zhang F, Xmonia X, Dong Z, Cui D. distribution of Cereal alloys in branched flour of the Yellow and Huai Valley of China and Science of non-hardness genes and of pigment in leather J.J.: J.S. J.A. J. sub.S. J. J.S. J. sub.S. J.A. J. A. B. A., 2010, 51: 235-237), the hardness and soft protein genotype of T5 DL.5 DS-2RS, Huizhou red, Yannong 19, Yangmai 158 and Zhegmai 9023 (publicly known and publicly used, wheat variety of China) are identified, and the loss of the T5 DL.5 DS-2RS hardness and soft protein genes is determined; meanwhile, specific amplification primers Xlfz6003 and Xlfz6004 (see the reference: Cao Zhen lan. accurate identification and effect analysis of wheat-rye small fragment translocation line T5 DL.5 DS-2RS [ D ]. Nanjing agriculture university Master academic thesis, 2018) designed by utilizing rye gamma-secalin subunit sequences identify T5 DL.5 DS-2RS and hybrid offspring thereof, determine that the T5 DL.5 DS-2RS carries secalin protein genes specific to rye, and find that the Secallin protein genes are stably expressed in the translocation line through alcohol soluble protein SDS-PAGE analysis.

Finally, the T5 DL.5 DS-2RS and hybrid progeny thereof (Yangmai 158/T5 DL.5 DS-2RS and Ninong 19/T5 DL.5 DS-2RS) are analyzed for hardness, protein content and agronomic characters, the main genetic effect of the translocation homozygote is determined, and the application potential in wheat quality breeding is determined.

Has the advantages that:

1. the invention discloses a breeding method of high-hardness wheat, the hardness of wheat grains bred by the method is obviously improved, the protein content and the lysine content are increased, the method is a new wheat quality breeding method, the genetic basis of the existing hard wheat can be greatly widened, a new common wheat variety with the grain hardness similar to that of the hard wheat is bred, and a new scheme is provided for improving the wheat quality;

2. the molecular marker combination disclosed by the invention can effectively identify T5 DL.5 DS-2RS homozygote, heterozygote and translocation-free type through one-time amplification, and provides a reliable tool for rapidly cultivating high-hardness wheat;

3. the high-hardness wheat variety cultured by the breeding method disclosed by the invention contains the small-fragment uncompensated translocation chromosome T5 DL.5 DS-2RS, is stable in genetic transmission and shows an obvious positive effect, and provides a new test material for creating, researching and applying the small-fragment uncompensated translocation chromosome.

4. The breeding method disclosed by the invention provides a new method for breeding high-quality bread and noodle wheat varieties, and the breeding and application of high-hardness wheat provides a new development business opportunity for manufacturing of high-hardness wheat flour processing equipment and flour production in China.

Description of the drawings

FIG. 1 wheat-rye translocation line T5DL & 5DS-2RS chromosome GISH/FISH analysis; red, green, blue signals

FIG. 2 cytological identification of T5 DL.5 DS-2RS translocation breakpoints; red, green, blue signals

FIG. 3T 5DL & 5DS-2RS molecular marker analysis involving 5DS breakpoints

a: 5DS and 2RS translocation breakpoint determination

b: 5DS specific molecular markers lfz7979 and lfz7980 amplification map

Lanes: m: DL 2000; 1: chinese spring; 2: n5AT 5D; 3: n5DT 5B; 4: t5 DL.5 DS-2 RS; arrows indicate specific amplification segments

c: 2RS specific molecular markers lfz7760, lfz7761, lfz7827 and lfz7845 amplification map

Lanes: m: DL 2000; 1: hui county red; 2: vitex rye; 3: DA 2R; 4: t5 DL.5 DS-2RS

FIG. 4: amplification of molecular marker combinations (Xlf7979+ Xlf7772) in F3 populations hybridized with T5 DL.5 DS-2RS, Yangmai 158

Lanes: m: DL 2000; 1: yangmai 158; 2: t5 DL.5 DS-2 RS; 3-36: yangmai 158/T5 DL.5 DS-2RS F3 generation

FIG. 5: yangmai 158/T5 DL.5 DS-2RS F3 plant chromosome GISH and FISH analysis, red, green and blue are ONPM #4(pAs1-1, pAs1-3, pAs1-4, pAs1-6, AFA-3, AFA-4, FAM modified pSc119.2-1, (GAA)₁₀Digoxigenin-11dUTP (dig) marking Jingzhou rye genome and blocking with Hui county red genome DNA

a: a T5 DL.5 DS-2RS homozygous translocation; b: t5 DL.5 DS-2RS hybrid translocation; c: no translocation, arrow indicates translocation chromosome

FIG. 6: pin gene specific primer amplification analysis

A: from left to right, the primers are as follows: Pina-D1, Pina-D1b, Tubulin;

b: from left to right, the primers are as follows: Pinb-D1, Pinb-D1a, Pinb-D1b, Tubulin;

c: from left to right, the primers are as follows: tubulin, Gsp-1, Gsp-D1;

lanes: m: DL 2000; 1: hui county red; 2: yangmai 158; 3: tobacco grower 19; 4: zheng Mai 9023; 5: t5 DL.5 DS-2RS

FIG. 7: SDS-PAGE analysis of the gamma-secalin protein

Lanes: m: DL 2000; 1: chinese spring; 2: vitex rye; 3: hui county red; 4: vitex rye; 5: jinghui No. 1; 6: DA 2R; 7: t5 DL.5 DS-2 RS; 8: DtA2RL

FIG. 8: gamma-secalin protein specific primer amplification analysis

a: xlfz 6003; b: xlfz 6004; c: tubulin specific primer

Lanes: m: DL 2000; 1: hui county red; 2: vitex rye; 3: jinghui No. 1; 4: DA 2R; 5: t5 DL.5 DS-2RS

b：Xlfz60041

Lanes: m: DL 2000; 1: yangmai 158; 2: t5 DL.5 DS-2 RS; 3-12: yangmai 158/T5 DL.5 DS-2RS F3 generation

Fifth, detailed description of the invention

1. Accurate identification of translocation breakpoint of wheat-Jingzhou rye translocation line T5 DL.5 DS-2RS

The line T5-852 translocation was performed using the rye Genome in situ hybridization, oligonucleotide probe set #4 (see references: Du P, Zhuang L, Wang Y, Yuan L, Wang Q, Wang D, Dawadontup, Tan L, Shen J, Xu H, ZHao H, Chu C, Qi Z.development of oligonucleotides and multiplex probes for quick and acid identification of wheat and wheat fibers) and oligonucleotide probe set #5 (see reference: Raynaud et. A. identification and application [ D ]. southern agricultural university, 2019., Symphasis) for simultaneous identification of wheat and rye chromosomes or wheat and rye chromosomes, and the translocation line T5-852 translocation line T5-352 translocation line (see GeneS.J.), the translocation chromosome is homozygous, the translocation breakpoint of the wheat chromosome is positioned at the near end of the 5D short arm, the translocation fragment of the rye chromosome is 2RS, the translocation segment accounts for about 40 percent of the short arm of 2R, the breakpoint is positioned at 2RS FL 0.6, and the breakpoint on the 5D is positioned in the AFA family signal region at the top end of 5DS, but the specific size is difficult to identify (figure 2). The annotated gene sequence of The wheat D genome (Triticum aestivum Ensembl plants, Release 30) was used to simultaneously amplify all The homologous regions of wheat for The gene sequence of rice for The gene sequence of wheat for The gene sequence of spring for China (TGACv1, https:// heat-urgent-users. versals. inra. fr/Seq-reproducibility/Assymbles) and for The gene sequence of rye (Bauer E, Schmutzer T, Barilar I, Mascher M, Guillach H, Martis 4M, Twdziok S, Hackauf B, Gordo A, Wilde P, Schmidt M, Korzun V, Mayer K, Schmid K, Schon C, Scholz U.Towards a w-genome for The gene of wheat for The gene of interest L.J.J.T.T. J. T.T. 2015, wherein The homologous regions of wheat for The gene sequence of spring for The gene sequence of China (SEQ ID No. 7, III) were amplified, SEQ ID No. 7, SEQ, in addition, only wheat specific bands can be amplified in 10 pairs, and the positions of the molecular markers on 2R are determined (Table 2; FIG. 3).

In order to clarify the breakpoint position of the translocation line on the 5D chromosome, using the position of the Pin gene, the gene sequence (Triticum aestivum Ensembl plants, Release 30) of about 1bp to 5Mb on 5D was retrieved, and compared with the genome reference sequence (TGACCV 1, https:// heat-urg. versals. inra. fr/Seq-reproducibility/Assembles) of Chinese spring A, B, 11 pairs of primers were designed, 8 pairs of primers amplified on wheat (Table 1), wherein primer lfz7979 amplified three bands of 5A, 5B and 5D in Chinese spring, and only two bands of 5A and 5B in both N5DT 5DT 34 and translocation line T5 DL. 5DS-2RS, but lacked a specific band on 5D, indicating that the segment of 5D with the marker is deleted, the same principle that lfz. 5DS-2RS is located on 5D chromosome, and the specific marker of N595D 3 lacks the marker segment (Table 3), in other materials, the amplification was stable and no deletion was present, so that the translocation breakpoint was located in the region of about 0.56Mb between 5D chromosomes 3752285-4310326, and the 5DS deletion region in the T5 DL.5 DS-2RS was about 3.8Mb, which contains a total of 96 genes, including the Pina, Pinb, and Gsp-1 genes (http:// 202.194.139.32/searchools /).

TABLE 12 primer sequences for R and 5D chromosome-specific molecular markers

2. Molecular marker combinatorial identification and molecular marker assisted selection following T5 DL.5 DS-2RS

Through the wheat-rye translocation line T5 DL.5 DS-2RS (see references: Zhuang L, Liu P, Liu Z, Chen T, Wu N, Sun L, Qi Z. multiple structural associations and physical mapping of eye chromosome 2R interregessed in o wheat)]Molecular Breeding, 2015, 35: 133-144.) are hybridized with tobacco grower 19, Yangmai 158 and Zhengmai 9023 (publicly known and publicly used, local wheat variety in China) and F is obtained by selfing₂And F₃And (4) seeds. In order to develop a specific molecular marker combination which can be amplified by one-time PCR and can simultaneously identify T5 DL.5 DS-2RS homozygote, heterozygote and translocation-free individual, the patent identifies an effective marker combination by repeated experiments, namely 2RS-1 is adoptedThe combination of the specific marker Xlfz7772 and the specific marker Xlfz7979 of 5DS can accurately identify three genotypes by one-time amplification (figure 4), cytogenetic identification is carried out on three genotype individuals identified by the molecular marker (figure 5), the effectiveness of the molecular marker combination is proved, and the two F3 population individuals are divided into three different types according to the effectiveness (Table 4). Statistical analysis shows that T5 DL.5 DS-2RS has reduced transmission frequency in hybrid offspring, but homozygote selfing offspring chromosome transmission is stable, which indicates that the translocation chromosome can be stably transmitted.

TABLE 22 primer amplification results of chromosome-specific molecular markers

TABLE 35 primer amplification results for chromosome-specific molecular markers

TABLE 4 translocation of chromosome T5 DL.5 DS-2RS at heterozygous F₃Transmission analysis of individual selfed progeny

Note: chi-square critical value² _0.05＝5.99

3. Pin, Gsp-1 and gamma-secalin gene amplification assay

To determine the hardness, soft protein and secalin protein in T5 DL.5 DS-2RS, the presence or absence of soft protein and secalin protein in rye was determined by using previously designed specific primers for the Pin and Gsp-1 genes (Table 5; see references: Chen F, Zhang F, Morris C, He Z, Xia X, Cui D. Molecular characterization of the pumoindoline a-D1b alloy and depth of anchors marker in wheat straw (Triticum aestivum L.) [ J ]. Journal of center Science 2010, 378 (1): 80-82; Chen F, Zhang F, Xia X, Dong Z, Cui D. distribution of pumoin in wheat straw of the Yellow and blood protein, Molecular analysis of wheat straw and wheat straw, cement J.: Molecular analysis of Molecular mineral J.371, branched peptide J.371, Molecular protein, Molecular analysis of Molecular protein, Molecular analysis of Molecular protein, Molecular analysis of Molecular protein of subunit of Molecular protein of. The clinical and Applied Genetics, 1997, 95 (5-6): 857-864; massa A N, Morris C F, Molecular evaluation of the puroindoline-a, puroindoline-b, and grain software protein-1 genes in the three diagnostic [ J ]. Journal of Molecular evaluation, 2006, 63 (4): 526-; meanwhile, Gsp-1 and GSP-D1 bands were amplified from Hui county red, Yannong 19, Yangmai 158 and Zheng wheat 9023, while only Gsp-1 band was amplified from T5 DL.5 DS-2RS, and GSP-D1 band was not amplified, indicating that the Gsp-1 gene on 5D was deleted (FIG. 6). Alcohol soluble protein SDS-PAGE amplification analysis was performed on Chinese spring, Huizhou red, Jingzhou rye (publicly known and publicly available, local variety of rye wheat in China), Jinghui No. 1 (Huizhou red-Jingzhou rye diploid), Huizhou red-Jingzhou rye dimer addition line DA2R (see reference: Lieky, Qi enhancement, Qiyouyou, etc., general wheat Huizhou red-Jingzhou rye heterostaining system breeding and its spindle mosaic disease resistance identification, journal of crops, 2007, 33 (4): 639 + 645) and T5 DL.5 DS-2RS, and it was found that JZHHM, JH1, DA2R and T5 DL.5 DS-2RS all have 75k gamma-secalin subunit (FIG. 7), while Chinese spring and Huizhou red do not. PCR amplification analysis was further performed on translocation lines and their hybrid progeny by using Secalin specific primers Xlfz6003 and Xlfz6004 (Table 5; see reference: Caoan. accurate identification and effect analysis of wheat-rye small fragment translocation line T5 DL.5 DS-2RS [ D ]. Nanjing university of agriculture university Master academic thesis, 2018.), which demonstrated that all translocation individuals and JZHHM, JH1 and DA2R all contained the same specific amplification product (FIG. 8), indicating that T5 DL.5 DS-2RS contained gamma-Secalin subunit-encoding genes and could be stably expressed in wheat background.

TABLE 5 primers for the Puroindoline-D1 and Secalin genes used in this study

4. T5 DL.5 DS-2RS quality and agronomic character analysis

The grain hardness is measured by a single grain determinator (Perten 4100) to find that the grain hardness (79.21) of T5 DL.5 DS-2RS is obviously increased compared with that of the parent Huizian red (59.97) (see the reference document: Cao Zhen lan. wheat-rye small fragment translocation line T5 DL.5 DS-2RS accurate identification and effect analysis [ D ] Nanjing agriculture university Master academic thesis, 2018.). For T5 DL.5 DS-2RS, Yangmai 158 and Nicotiana 19 (two varieties are hard wheat) hybrid F3 populations are divided into homozygous translocation and non-translocation individuals according to molecular marker analysis, 30 individuals are respectively and randomly determined for each type, and the results show that the average grain hardness of the homozygous translocation population is remarkably higher than that of the non-translocation population, the grain hardness is respectively improved by 11.70 and 16.34 compared with the non-translocation population, the difference is remarkable (Table 6), and the introduction of T5 DL.5 DS-2RS can remarkably improve the grain hardness, and the method is an important gene resource for breeding high-hardness wheat. For two T5 DL.5 DS-2RS and Yangmai 158, two hybrid F3 populations of Yannong 19, the protein content in the two populations of seeds is measured by a near infrared tester, and the homozygous translocation population protein content is found to be obviously increased, wherein the T5 DL.5 DS-2RS and Yangmai 158 populations reach a significant level (Table 7). The agronomic character comparison shows that except the difference of the spike numbers of the single plants in Yangmai 158 and T5 DL.5 DS-2RS populations, other characters of the homozygous translocation population have no significant difference with the translocation-free population (Table 8), and the T5 DL.5 DS-2RS has no obvious adverse effect.

TABLE 6 comparison of kernel hardness of T5 DL.5 DS-2RS and cultivars and their F3 mixed isolates

Note:^*indicates significance P < 0.05

TABLE 7T 5DL & 5DS-2RS and cultivars and F thereof₃Population mean protein content t test

Note:^*indicates significance P < 0.05

TABLE 8 comparison of agronomic traits in T5 DL.5 DS-2RS and cultivar hybrid F3 segregating populations

Note: "b" indicates that the difference was up to a 5% significant level.

Claims

1. A breeding method for breeding high-hardness wheat comprises the following steps:

(1) adopting a wheat-Jingzhou rye small-fragment translocation line T5 DL.5 DS-2RS as a donor, hybridizing with a cultivated wheat variety with excellent properties to obtain a hybrid F1, and selfing to obtain F2 and F3 seeds;

(2) carrying out amplification analysis on F2 and F3 plants by utilizing a marker combination (Xlfz7772 and Xlfz7979) to identify the chromosome composition, determining T5 DL.5 DS-2RS homozygote and heterozygote, and breeding T5 DL.5 DS-2RS homozygote with excellent properties by combining phenotypic analysis;

(3) carrying out chromosome fluorescence in situ hybridization analysis on the T5 DL.5 DS-2RS homozygote identified by the molecular marker to determine a translocation chromosome;

(4) and (3) carrying out hardness determination on the T5 DL.5 DS-2RS homozygote with accurate identification, identifying the hardness index of the homozygote, and breeding a high-hardness excellent strain through continuous selfing and hardness determination.

2. 2 pairs of amplification primers, mixed together following the translocation line according to claim 1, can simultaneously amplify specific fragments of rye 2RS and 5DS, recognizing T5 DL.5 DS-2RS homozygotes, heterozygotes and translocation-free individuals, and having the following sequence characteristics:

3. use of the breeding method according to claim 1 and the amplification primer pair according to claim 2 in commercial breeding of wheat.