A substance, method and application for detecting multiple insertion / deletion polymorphism of Leymus chinensis

Abstract

The present invention discloses a substance, method, and application for detecting multiple insertion / deletion polymorphisms in Leymus chinensis, belonging to the technical field of assays or detection methods involving enzymes, nucleic acids, or microorganisms. This application can be used to identify or assist in the identification of Leymus chinensis fruit set rate using 30 primer pairs, from InDel-1-P to InDel-30-P. These 30 primer pairs can accurately and efficiently identify varieties with this trait, facilitating the identification and screening of high-fruit-set Leymus chinensis germplasm during the seedling stage by breeders, accelerating the selection of new Leymus chinensis varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of determination or detection methods involving enzymes, nucleic acids or microorganisms, and particularly relates to a substance, method and application thereof for detecting multiple insertion / deletion polymorphisms of Leymus chinensis. Background Art

[0002] Molecular markers can be applied to plant germplasm and variety identification, as well as transgenic identification. DNA molecular identification is a new biological identification system created by utilizing the specificity of standard, sufficiently variable, easily amplified, and relatively short DNA fragments within a species or the diversity between species. It can quickly and automatically identify species or samples.

[0003] Leymus chinensis, also known as alkali grass, is a perennial rhizomatous plant belonging to the genus Leymus in the Poaceae family. It is primarily distributed in my country's three northeastern provinces and the Inner Mongolia Autonomous Region, and is also found in Russia, Japan, and North Korea. It is a key community in the meadow steppes and arid steppes of the eastern Eurasian steppe region. Leymus chinensis boasts excellent quality, high nutritional value, strong stress tolerance, and good palatability, making it a key component in the development of artificial grasslands and the improvement of degraded grasslands. However, in its natural habitat, asexual reproduction is dominant, while sexual reproduction is relatively weak, manifesting in three key issues: a low seed set rate of approximately 25%, a low heading rate of approximately 7.9%, and a low seed germination rate of 10%-20%. This low sexual reproduction capacity poses significant challenges to the practical production and application of Leymus chinensis, severely limiting its large-scale artificial cultivation. In industrialization, a plant's ability to reproduce seeds determines its practical application potential. Only forage grasses with high seed reproduction coefficients can be widely used. However, the low fruit set rate and germination rate of Leymus chinensis, as well as its long breeding cycle, have long restricted the development and utilization of new varieties. Given the rapid development of animal husbandry and the huge demand for ecological restoration (construction), the selection and breeding of new Leymus chinensis varieties with high fruit set rates is urgent.

[0004] However, because Leymus chinensis is a perennial plant, traditional hybridization breeding techniques require a long cycle. It takes two to three years for flowering and fruiting to determine the fruit set rate of candidate materials created through hybridization. This allows for the screening of new Leymus chinensis germplasm with high fruit set rates and the breeding of new varieties. Therefore, there is an urgent need to develop molecular marker combinations that can be used for rapid and efficient screening at the seedling stage, and to establish corresponding low-cost, highly efficient, and accurate application processes.

[0005] The seed setting trait of Leymus chinensis is a sexual reproductive trait controlled by multiple genes. Conventional single SNP markers are difficult to accurately distinguish between Leymus chinensis germplasms with different seed setting rates. Using molecular marker technology to analyze the genetic diversity and phylogenetic relationships of Leymus chinensis germplasm resources is fundamental to elucidating the genetic basis of Leymus chinensis and improving its germplasm resources. Molecular marker technology has developed rapidly in recent years. However, due to the large genome size, complex genetic background, and high proportion of repetitive sequences in Leymus chinensis, there are currently no molecular markers that cover the entire genome and can be used to accurately identify the genetic diversity of Leymus chinensis seed setting. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a substance capable of detecting and identifying the fruit set rate of Leymus chinensis. The technical problem to be solved is not limited to the technical subject described above, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides an application of a substance for detecting multiple insertion / deletion polymorphism of Leymus chinensis in identifying or assisting in identifying the fruit setting rate of Leymus chinensis (identifying or assisting in identifying the fruit setting rate of Leymus chinensis).

[0009] The substances are respectively named InDel-1-P, InDel-2-P, InDel-3-P, InDel-4-P, InDel-5-P, InDel-6-P, InDel-7-P, InDel-8-P, InDel-9-P, InDel-10-P, InDel-11-P, InDel-12-P, InDel-13-P, InDel4-1-P, InDel-15-P, InDel-1-P, I a composition consisting of these 30 primer pairs: InDel-16-P, InDel-17-P, InDel-18-P, InDel-19-P, InDel-20-P, InDel-21-P, InDel-22-P, InDel-23-P, InDel-24-P, InDel-25-P, InDel-26-P, InDel-27-P, InDel-28-P, InDel-29-P, and InDel-30-P;

[0010] The InDel-1-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 1 and SEQ ID No. 2; the InDel-2-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 3 and SEQ ID No. 4; the InDel-3-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 5 and SEQ ID No. 6; the InDel-4-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 7 and SEQ ID No. 8; the InDel-5-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 9 and SEQ ID No. 10; the InDel-6-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 11 and SEQ ID No. 12; the InDel-7-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 13 and SEQ ID No. 14; the InDel-8-P comprises two single-stranded DNAs having nucleotide sequences of SEQ ID No. 15 and SEQ ID No. The InDel-9-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 17 and SEQ ID No. 18; the InDel-10-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 19 and SEQ ID No. 20; the InDel-11-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 21 and SEQ ID No. 22; the InDel-12-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 23 and SEQ ID No. 24; the InDel-13-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 25 and SEQ ID No. 26; the InDel-14-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 27 and SEQ ID No. 28; the InDel-15-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 29 and SEQ ID No. 30; the InDel-16-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 31 and SEQ ID No. 32; the InDel-17-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 33 and SEQ ID No. 34; the InDel-18-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 35 and SEQ ID No. 36; the InDel-19-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No.The InDel-20-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 39 and SEQ ID No. 40; the InDel-21-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 41 and SEQ ID No. 42; the InDel-22-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 43 and SEQ ID No. 44; the InDel-23-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 45 and SEQ ID No. 46; the InDel-24-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 47 and SEQ ID No. 48; the InDel-25-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 49 and SEQ ID No. 50; the InDel-26-P is composed of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 51 and SEQ ID No. The InDel-27-P comprises two single-stranded DNAs with nucleotide sequences of SEQ ID No. 53 and SEQ ID No. 54; the InDel-28-P comprises two single-stranded DNAs with nucleotide sequences of SEQ ID No. 55 and SEQ ID No. 56; the InDel-29-P comprises two single-stranded DNAs with nucleotide sequences of SEQ ID No. 57 and SEQ ID No. 58; and the InDel-30-P comprises two single-stranded DNAs with nucleotide sequences of SEQ ID No. 59 and SEQ ID No. 60.

[0011] The InDel-1-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_1 of Leymus chinensis. The InDel_1 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism thereof is a nucleotide sequence of 5′-CTGT-3′ at positions 236115390 to 236115393 of the Lc1Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence at this position in high-fruiting materials is 5′-CT-3′, and the nucleotide sequence at this position in low-fruiting materials is 5′-CTGT-3′.

[0012] The InDel-2-P is a polymorphic primer pair for detecting the InDel molecular marker of Leymus chinensis named InDel_2, wherein the InDel_2 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof is 5′-TAG-3′ at positions 261464410 to 261464412 of the Lc1Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence at this position in high-fruiting materials is 5′-T-3′, and the nucleotide sequence at this position in low-fruiting materials is 5′-TAG-3′;

[0013] The InDel-3-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_3 of Leymus chinensis. The InDel_3 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 295721606 to 295721611 of the Lc1Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CGCGAG-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-C-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CGCGAG-3′.

[0014] The InDel-4-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_4 of Leymus chinensis. The InDel_4 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 505186724 to 505186727 of the Lc2Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-ATAG-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-ATAG-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-A-3′.

[0015] The InDel-5-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_6 of Leymus chinensis. The InDel_5 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 505186723 to 505186726 of the Lc2Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CATA-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-CATA-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-C-3′.

[0016] The InDel-6-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_6 of Leymus chinensis. The InDel_6 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence at position 567528306 of the Lc2Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-T-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-T-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-TCG-3′.

[0017] The InDel-7-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_7 of Leymus chinensis. The InDel_7 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 567528307 to 567528308 of the Lc2Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-TG-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-TG-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-TGTCG-3′.

[0018] The InDel-8-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_3 of Leymus chinensis. The InDel_3 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence at position 156615192 of the Lc3Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence at this position is 5′-G-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-G-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-GAA-3′.

[0019] The InDel-9-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_9 of Leymus chinensis. The InDel_9 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 200471157 to 200471162 of the Lc3Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CGTGTT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-C-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CGTGTT-3′.

[0020] The InDel-10-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_10, wherein the InDel_10 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence of positions 471241350 to 471241357 of the Lc4Ns chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-GCCCCCCC-3′, the nucleotide sequence at this position of the high-fruiting material is 5′-GCCCCCCC-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-GC-3′;

[0021] The InDel-11-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_11 of Leymus chinensis. The InDel_11 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 471241352 to 471241353 of the Lc4Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-TC-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-TCCCCCGC-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-TC-3′.

[0022] The InDel-12-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_12, wherein the InDel_12 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence at positions 254641576 to 254641578 of the Lc4Xm chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-CTT-3′, the nucleotide sequence at this position of the high-fruiting material is 5′-CTTGTT-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CTT-3′;

[0023] The InDel-13-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_13 of Leymus chinensis. The InDel_13 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 289253984 to 289253985 of the Lc4Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-TA-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-TCTAA-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-TAA-3′.

[0024] The InDel-14-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_14 of Leymus chinensis. The InDel_14 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 58489729 to 58489737 of the Lc5Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-ATTTGCATT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-A-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-ATTTGCATT-3′.

[0025] The InDel-15-P is a polymorphic primer pair for detecting the InDel molecular marker of Leymus chinensis named InDel_15, wherein InDel_15 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence of positions 264968596 to 264968599 of the Lc5Xm chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-CAGA-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-CAGA-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-C-3′.

[0026] The InDel-16-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_16. The InDel_16 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 316997496 to 316997497 of the Lc5Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-TT-3′. The nucleotide sequence at this position in high-fruiting materials is 5′-TT-3′, and the nucleotide sequence at this position in low-fruiting materials is 5′-TTCTCT-3′.

[0027] The InDel-17-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_17 of Leymus chinensis. The InDel_17 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 409910944 to 409910947 of the Lc5Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-GTTT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-GTTT-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-GT-3′.

[0028] The InDel-18-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_18, wherein the InDel_18 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence at positions 415583994 to 415583999 of the Lc5Xm chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-AAAGGT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-AAAGGT-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-A-3′.

[0029] The InDel-19-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_19 of Leymus chinensis. The InDel_19 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 288915855 to 288915863 of the Lc6Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-GAAACCAAA-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-G-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-GAAACCAAA-3′.

[0030] The InDel-20-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_20 of Leymus chinensis. The InDel_20 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence at position 457382318 of the Lc6Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-C-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-C-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CTCA-3′.

[0031] The InDel-21-P is a polymorphic primer pair for detecting the InDel molecular marker of Leymus chinensis named InDel_21, wherein the InDel_21 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence of positions 89988661 to 89988663 of the Lc7Ns chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-AAG-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-AAG-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-A-3′.

[0032] The InDel-22-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_22, wherein the InDel_22 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence at positions 373098439 to 373098443 of the Lc7Ns chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-CTTTT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-CTT-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CTTTT-3′.

[0033] The InDel-23-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_23, wherein the InDel_23 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis, and the polymorphism thereof corresponds to the nucleotide sequence at positions 379567923 to 379567926 of the Lc7Ns chromosome of the Leymus chinensis reference genome, wherein the nucleotide sequence is 5′-ACCC-3′, the nucleotide sequence at this position of the high-fruiting material is 5′-ACCC-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-ACCCCCC-3′;

[0034] The InDel-24-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_24 of Leymus chinensis. The InDel_24 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 379567924 to 379567927 of the Lc7Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CCCG-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-CCCG-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CCCGCCG-3′.

[0035] The InDel-25-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_25 of Leymus chinensis. The InDel_25 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 441863628 to 441863629 of the Lc7Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CT-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-CTGT-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CT-3′.

[0036] The InDel-26-P is a polymorphic primer pair for detecting the Leymus chinensis InDel molecular marker named InDel_26. The InDel_26 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 556564709 to 556564712 of the Lc7Ns chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-CCAG-3′. The nucleotide sequence at this position in high-fruiting materials is 5′-C-3′, and the nucleotide sequence at this position in low-fruiting materials is 5′-CCAG-3′.

[0037] The InDel-27-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_27 of Leymus chinensis. The InDel_27 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 525649946 to 525649950 of the Lc7Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-GCAAC-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-GCAAC-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-G-3′.

[0038] The InDel-28-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_28 of Leymus chinensis. The InDel_28 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence of positions 525653482 to 525653485 of the Lc7Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-TGCA-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-TGCAGCA-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CTGT-3′.

[0039] The InDel-29-P is a polymorphic primer pair for detecting the InDel molecular marker named InDel_29 of Leymus chinensis. The InDel_29 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. The polymorphism corresponds to the nucleotide sequence at position 538134266 of the Lc7Xm chromosome of the Leymus chinensis reference genome. The nucleotide sequence is 5′-C-3′. The nucleotide sequence at this position of the high-fruiting material is 5′-C-3′, and the nucleotide sequence at this position of the low-fruiting material is 5′-CCCCAAT-3′.

[0040] The InDel-30-P is a polymorphic primer pair for detecting the InDel molecular marker of Leymus chinensis named InDel_30. The InDel_30 is an insertion-deletion marker on the Lc1Ns chromosome of Leymus chinensis. Its polymorphism corresponds to the nucleotide sequence at position 567319996 of the Lc7Xm chromosome of the Leymus chinensis reference genome. Its nucleotide sequence is 5′-C-3′. The nucleotide sequence at this position of the high-fruiting material is: 5′-C-3′, and the nucleotide sequence at this position of the low-fruiting material is or 5′-CGTT-3′.

[0041] The present invention also provides a composition for identifying or assisting in identifying the fruit setting rate of Leymus chinensis (identifying or assisting in identifying the fruit setting rate of Leymus chinensis), and the composition is the aforementioned substance.

[0042] The present invention also provides a method for identifying or assisting in identifying the fruit setting rate of Leymus chinensis, the method comprising the following steps: using the genomic DNA of the Leymus chinensis to be identified as a template, performing PCR amplification using each primer pair in the aforementioned composition to obtain PCR products of 30 primer pairs, and identifying the fruit setting rate of Leymus chinensis according to whether the PCR products of the 30 primer pairs have specific bands, the fruit setting rate of Leymus chinensis with specific bands of the PCR products of the 30 primer pairs is higher than that of Leymus chinensis with no specific bands of the PCR products of the 30 primer pairs, and the presence of specific bands in the PCR products of the 30 primer pairs satisfies the following 30 conditions:

[0043] 1) The PCR product of the InDel-1-P primer pair contains a band of 750-2000 bp; 2) The PCR product of the InDel-2-P primer pair contains a band of 750-2000 bp; 3) The PCR product of the InDel-3-P primer pair contains a band of 750-2000 bp; 4) The PCR product of the InDel-4-P primer pair contains a band of 750-2000 bp; 5) The PCR product of the InDel-5-P primer pair contains a band of 750-2000 bp; 6) The PCR product of the InDel-6-P primer pair contains a band of 750-2000 bp; 7) The PCR product of the InDel-7-P primer pair contains a band of 750-2000 bp. 2000 bp band; 8) The PCR product of the InDel-8-P primer pair contains a 750-2000 bp band; 9) The PCR product of the InDel-9-P primer pair contains a 750-2000 bp band; 10) The PCR product of the InDel-10-P primer pair contains a 500-2000 bp band; 11) The PCR product of the InDel-11-P primer pair contains a 500-2000 bp band; 12) The PCR product of the InDel-12-P primer pair contains a 500-2000 bp band; 13) The PCR product of the InDel-13-P primer pair contains a 500-2000 bp band; 14) The InDel- The PCR product of the InDel-14-P primer pair contains a band of 500-2000 bp; 15) the PCR product of the InDel-15-P primer pair contains a band of 750-2000 bp; 16) the PCR product of the InDel-16-P primer pair contains a band of 750-2000 bp; 17) the PCR product of the InDel-17-P primer pair contains a band of 750-2000 bp; 18) the PCR product of the InDel-18-P primer pair contains a band of 750-2000 bp; 19) the PCR product of the InDel-19-P primer pair contains a band of 750-2000 bp; 20) the PCR product of the InDel-20-P primer pair contains a band of The PCR product of the InDel-21-P primer pair contains a band of 750-2000 bp; 21) The PCR product of the InDel-21-P primer pair contains a band of 750-2000 bp; 22) The PCR product of the InDel-22-P primer pair contains a band of 750-2000 bp; 23) The PCR product of the InDel-23-P primer pair contains a band of 750-2000 bp; 24) The PCR product of the InDel-24-P primer pair contains a band of 750-2000 bp; 25) The PCR product of the InDel-25-P primer pair contains a band of 750-2000 bp; 26) The PCR product of the InDel-26-P primer pair contains a band of 750-2000 bp;27) The PCR product of the InDel-27-P primer pair contained a band of 750-2000 bp; 28) The PCR product of the InDel-28-P primer pair contained a band of 750-2000 bp; 29) The PCR product of the InDel-29-P primer pair contained a band of 750-2000 bp; 30) The PCR product of the InDel-30-P primer pair contained a band of 750-2000 bp.

[0044] The PCR products of the 30 primer pairs have no specific bands, which means that at least one of the above 30 conditions is not met.

[0045] In the above method, the annealing condition in the PCR amplification cycle is annealing at 55° C. for 30 seconds.

[0046] In the above method, the extension condition in the PCR amplification cycle is 72° C. for 60 s.

[0047] The present invention also provides the use of the above-mentioned substance in identifying or assisting in identifying whether the seedlings of Leymus chinensis are high-fruiting-rate germplasm materials or varieties.

[0048] The present invention also provides a product for identifying or assisting in identifying whether the aforementioned substance is a germplasm material or variety with a high fruit-setting rate during the seedling stage of preparing Leymus chinensis.

[0049] The present invention also provides the use of the above-mentioned substance in identifying or assisting in identifying whether the seedlings of Leymus chinensis are germplasm materials or varieties with low fruit setting rates.

[0050] The present invention also provides the use of the above-mentioned substance in preparing a product for identifying or assisting in identifying whether a germplasm material or variety of Leymus chinensis has a low fruit-setting rate during the seedling stage.

[0051] The present invention also provides application of the above-mentioned substance in Leymus chinensis breeding or products of Leymus chinensis breeding.

[0052] The seedling stage is the stage when the 3 true leaves of the chinensis seeds germinate. The low fruit set rate is a fruit set rate lower than 10%. The high fruit set rate is a fruit set rate higher than 65%.

[0053] The product may be a kit comprising primers for detecting the InDel molecular marker combination. The kit of the present invention may also include other reagents for PCR amplification. The other reagents for PCR amplification may include Buffer, dNTPs, Taq enzyme, and ddH2O.

[0054] The present invention also provides a method for breeding Leymus chinensis, comprising using the genomic DNA of the parent Leymus chinensis to be selected as a template, performing PCR amplification using each primer pair in the aforementioned composition, obtaining PCR products of 30 primer pairs, and selecting Leymus chinensis with specific bands in the PCR products of the 30 primer pairs as parents for breeding, wherein the specific bands in the PCR products of the 30 primer pairs meet the following 30 conditions:

[0055] 1) The PCR product of the InDel-1-P primer pair contains a band of 750-2000 bp; 2) The PCR product of the InDel-2-P primer pair contains a band of 750-2000 bp; 3) The PCR product of the InDel-3-P primer pair contains a band of 750-2000 bp; 4) The PCR product of the InDel-4-P primer pair contains a band of 750-2000 bp; 5) The PCR product of the InDel-5-P primer pair contains a band of 750-2000 bp; 6) The PCR product of the InDel-6-P primer pair contains a band of 750-2000 bp; 7) The PCR product of the InDel-7-P primer pair contains a band of 750-2000 bp. -2000 bp band; 8) The PCR product of the InDel-8-P primer pair contains a 750-2000 bp band; 9) The PCR product of the InDel-9-P primer pair contains a 750-2000 bp band; 10) The PCR product of the InDel-10-P primer pair contains a 500-2000 bp band; 11) The PCR product of the InDel-11-P primer pair contains a 500-2000 bp band; 12) The PCR product of the InDel-12-P primer pair contains a 500-2000 bp band; 13) The PCR product of the InDel-13-P primer pair contains a 500-2000 bp band; 14) The InDel The PCR product of the InDel-14-P primer pair contains a band of 500-2000 bp; 15) the PCR product of the InDel-15-P primer pair contains a band of 750-2000 bp; 16) the PCR product of the InDel-16-P primer pair contains a band of 750-2000 bp; 17) the PCR product of the InDel-17-P primer pair contains a band of 750-2000 bp; 18) the PCR product of the InDel-18-P primer pair contains a band of 750-2000 bp; 19) the PCR product of the InDel-19-P primer pair contains a band of 750-2000 bp; 20) the PCR product of the InDel-20-P primer pair contains a band of The PCR product of the InDel-21-P primer pair contains a band of 750-2000 bp; 21) The PCR product of the InDel-21-P primer pair contains a band of 750-2000 bp; 22) The PCR product of the InDel-22-P primer pair contains a band of 750-2000 bp; 23) The PCR product of the InDel-23-P primer pair contains a band of 750-2000 bp; 24) The PCR product of the InDel-24-P primer pair contains a band of 750-2000 bp; 25) The PCR product of the InDel-25-P primer pair contains a band of 750-2000 bp; 26) The PCR product of the InDel-26-P primer pair contains a band of 750-2000 bp;27) The PCR product of the InDel-27-P primer pair contains a band of 750-2000 bp; 28) The PCR product of the InDel-28-P primer pair contains a band of 750-2000 bp; 29) The PCR product of the InDel-29-P primer pair contains a band of 750-2000 bp; 30) The PCR product of the InDel-30-P primer pair contains a band of 750-2000 bp.

[0056] The above method comprises the following steps: (1) extracting DNA of the leopard grass material to be identified; (2) performing PCR amplification on the target segment using a primer pair combination formed by a MIP molecular marker combination; (3) performing electrophoresis detection on the amplified product of the deep sequencing segment; (4) comparing the electrophoresis pattern of all InDel sites of the leopard grass material to be identified with the pre-established MIP amplification electrophoresis pattern of high-fruiting rate and low-fruiting rate leopard grass materials, thereby determining the type of fruiting rate of the material. Judgment rules:

[0057] ① High-fruiting-rate materials: Using the genomic DNA of the test sample as a template, if the primer pairs for all 30 InDel markers (InDel_1 to InDel_30) can amplify DNA fragments, the test material is considered to have a high-fruiting-rate material. ② Non-high-fruiting-rate materials: Using the genomic DNA of the test sample as a template, if the primer pairs for any of the 30 InDel markers (InDel_1 to InDel_30) fail to amplify DNA fragments, the test material is considered to have a non-high-fruiting-rate material.

[0058] In the above-described method, the PCR reaction system is as follows (total volume 25 μL): 1 μL DNA template, 1 μL forward primer and 1 μL reverse primer each, 2.5 μL 10× Buffer, 2 μL dNTP Mixture (10 mmol / L each), 0.25 μL Taq enzyme (20 U / μL), and 12.25 μL ddH2O. The final concentrations of the forward and reverse primers in the reaction system are both 5 μM. The PCR reaction conditions are as follows: initial denaturation at 94°C for 5 min; 36 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s; and extension at 72°C for 10 min.

[0059] The present invention has the advantages that, when using the Leymus chinensis InDel molecular marker combination of the present invention to identify the fruit set rate traits of Leymus chinensis, based on PCR technology, compared with other technologies, it has the characteristics of wide adaptability of the detection platform, easy operation, labeling flexibility, detection efficiency and the additivity of information, and has broad application prospects. In the face of the complexity of the Leymus chinensis genome, the present invention has finally obtained a relatively small number of, low-cost InDel molecular marker combinations through a large number of experiments and comparative analysis verifications, and this molecular marker combination can achieve accurate and efficient identification of Leymus chinensis fruit set rate trait varieties, which is conducive to breeders for carrying out identification and screening of high fruit set rate Leymus chinensis germplasm at the seedling stage, accelerating the breeding of new varieties of Leymus chinensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the distribution map of the identified SNP / Indel sites on the chromosomes of Leymus chinensis.

[0061] Figure 2 Statistics of the number of SNPs / Indels identified in some samples.

[0062] Figure 3 Statistics of SNP / Indel types identified in some samples.

[0063] Figure 4 The chromosome distribution and sequence information of the variant sites included in the MIP marker.

[0064] Figure 5 The results of PCR amplification of Leymus chinensis accessions T1-T4 using MIP markers. From left to right, they are Leymus chinensis accessions T1, T2, T3, and T4.

[0065] Figure 6 The results of PCR amplification of Leymus chinensis accessions T5-T8 using MIP markers. From left to right, they are Leymus chinensis accessions T5, T6, T7, and T8.

[0066] Figure 7 The results of PCR amplification of Leymus chinensis materials T9-T12 using MIP markers are shown in Figure 2.

[0067] Figure 8 The results of PCR amplification of Leymus chinensis materials T13-T16 using MIP markers are shown in Figure 2.

[0068] Figure 9 The results of PCR amplification of Leymus chinensis materials T17-T20 using MIP markers are shown in Figure 2.

[0069] Figure 10 The results of PCR amplification of Leymus chinensis materials T21-T24 using MIP markers are shown in Figure 2.

[0070] Figure 11 The results of PCR amplification of Leymus chinensis materials T25-T27 using MIP markers are shown.

[0071] Figure 12 The results of PCR amplification of Leymus chinensis materials T28-T30 using MIP markers are shown in Figure 2. DETAILED DESCRIPTION

[0072] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0073] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0074] Example 1. Development of molecular markers for identifying seed-setting rate types of Leymus chinensis at the seedling stage and design of specific amplification primer pairs

[0075] 1. MIP molecular marker combination development process

[0076] 1) Sixteen samples of Leymus chinensis leaves with low seed set (0 to 68%) were selected, with three individual spikes from each sample used as replicates. The leaves were ground with liquid nitrogen, and total RNA was extracted using the AllPure DNA / RNA / Protein Kit.

[0077] 2) A high-throughput sequencing library was constructed using the NEB transcriptome library kit.

[0078] 3) Using Illumina Hiseq TM Perform sequencing.

[0079] 4) After sequencing, the raw data were first quality-controlled, including removal of adapters and low-quality reads. The clean reads were then aligned to the Leymus chinensis reference genome (https: / / doi.org / 10.6084 / m9.figshare.24032238.v1) using BWAmem software. Reads that aligned to unique positions on the reference genome were screened for subsequent variant detection. SNPs and indels were screened using GATK software, with a minimum sequencing depth of at least 5x for SNP and indel sites.

[0080] 5) INDEL marker screening: The sequencing depth of INDEL markers used for MIP molecular marker development was no less than 2x; the detection rate of a single INDEL locus across samples was 100%; the minimum allele mutation frequency (MAF) was greater than 0.01; the distribution of the screened INDEL loci was relatively uniform; the screened INDEL loci had at least 100 bp upstream and downstream and no introns, and the screened loci differed only between the two materials with a fruit set rate of 0 and a fruit set rate of 68%; INDEL loci that met the above conditions could be merged, and the combination of loci that met the above conditions was used as the final MIP molecular marker set (Table 1).

[0081] Table 1. MIP marker combinations

[0082]

[0083]

[0084] 2. Specific primer pairs

[0085] Based on the MIP location information obtained in step 1 and referring to the Leymus chinensis genome sequence, a primer pair for specific amplification was designed. The primer sequences are as follows:

[0086] The primer pair InDel_1 consists of a forward primer of SEQ ID No. 1 and a reverse primer of SEQ ID No. 2, InDel_2 consists of a forward primer of SEQ ID No. 3 and a reverse primer of SEQ ID No. 4, InDel_3 consists of a forward primer of SEQ ID No. 5 and a reverse primer of SEQ ID No. 6, InDel_4 consists of a forward primer of SEQ ID No. 7 and a reverse primer of SEQ ID No. 8, InDel_5 consists of a forward primer of SEQ ID No. 9 and a reverse primer of SEQ ID No. 10, InDel_6 consists of a forward primer of SEQ ID No. 11 and a reverse primer of SEQ ID No. 12, InDel_7 consists of a forward primer of SEQ ID No. 13 and a reverse primer of SEQ ID No. 14, InDel_8 consists of a forward primer of SEQ ID No. 15 and a reverse primer of SEQ ID No. 16, and InDel_9 consists of a forward primer of SEQ ID No. 17 and a reverse primer of SEQ ID No. InDel_15 consists of a forward primer of SEQ ID No. 29 and a reverse primer of SEQ ID No. 30, InDel_16 consists of a forward primer of SEQ ID No. 31 and a reverse primer of SEQ ID No. 32, InDel_17 consists of a forward primer of SEQ ID No. 33 and a reverse primer of SEQ ID No. 34. No.34, InDel_18 consists of a forward primer of SEQ ID No.35 and a reverse primer of SEQ ID No.36, InDel_19 consists of a forward primer of SEQ ID No.37 and a reverse primer of SEQ ID No.38, InDel_20 consists of a forward primer of SEQ ID No.39 and a reverse primer of SEQ ID No.40, InDel_21 consists of a forward primer of SEQ ID No.41 and a reverse primer of SEQ ID No.42, InDel_22 consists of a forward primer of SEQ ID No.43 and a reverse primer of SEQ ID No.44, InDel_23 consists of a forward primer of SEQ ID No.InDel_28 consists of a forward primer SEQ ID No. 55 and a reverse primer SEQ ID No. 56, InDel_29 consists of a forward primer SEQ ID No. 57 and a reverse primer SEQ ID No. 58, and InDel_30 consists of a forward primer SEQ ID No. 59 and a reverse primer SEQ ID No. 60.

[0087] 3. Results and Analysis

[0088] 1) Sequencing data statistics and quality control

[0089] A total of 48 sequencing libraries were constructed for this sequencing run. Based on statistical data such as the number of high-quality reads and base error rate from each library, combined with data quality control analysis, the base quality (Q30) was above 90%, indicating good sequencing quality. A total of 354.85GB of data was generated from the 48 test samples, with an average of 7.39GB of valid data per sample. The alignment rate to the reference genome data ranged from 85.19% to 92.78%, indicating that the sequenced fragments covered a wide range of the reference genome and a high number of detectable variant sites. This also demonstrates the high randomness of the sequencing, making it suitable for subsequent marker development needs.

[0090] 2) Mutation Detection

[0091] By comparing the sequencing data to the reference genome, a total of 1,488,272 SNP / Indel sites were detected in 48 samples ( Figure 1 and Figure 2 ), of which 588,371 were transition SNPs, 784,853 were transversion SNPs, and 115,048 were indels. These variant sites were mainly distributed in intergenic regions, intronic regions, 2Kbp upstream and 2Kbp downstream of genes, splice sites, and within 2Kbp upstream and downstream of the overlap of two genes. The largest number of SNP sites were located in intergenic regions ( Figure 3). Marker development. This invention develops MIP molecular markers for identifying the fruit set trait in Leymus chinensis for the first time, with the aim of achieving accurate identification of Leymus chinensis resources in the seedling stage. According to the principle of uniform distribution of indel markers on chromosomes, a set of MIP markers containing 30 indel sites were analyzed, screened, and combined among numerous INDEL sites to develop a set of MIP markers (Table 1). Site-specific analysis found that the 30 indel sites contained in the MIP markers developed by the present invention can distinguish samples with a fruit set rate lower than 10% from samples with a fruit set rate higher than 65% ( Figure 4 ), which shows that the MIP marker combination developed in the present invention has a strong ability to identify the fruit-bearing traits of Leymus chinensis.

[0092] Example 2: Design of primer pairs for specific detection based on the developed MIP marker combination

[0093] In this example, the specificity of the developed labeled primer pair was verified by a double-blind test.

[0094] 1. Extraction of DNA from plant materials

[0095] In the experimental field, 30 Leymus chinensis germplasm materials (labeled as T1-T30) were selected to extract total DNA from the leaves at the budding stage, and the extracted total DNA was subjected to 1% agarose gel electrophoresis. The agarose gel electrophoresis results of the total DNA of T1-T30 were as follows: Figure 5-12 The results showed that the extracted DNA had obvious electrophoresis bands larger than 20kb, indicating that high-purity and relatively complete DNA was obtained.

[0096] 2. PCR amplification

[0097] Using the total DNA sample extracted in step 1 as a template, PCR amplification was performed using the primer pairs designed in step 2 of Example 1 to obtain PCR products. The PCR reaction system was as follows (total volume 25 μL): 1 μL DNA template, 1 μL each of F and R primers, 2.5 μL 10× Buffer, 2 μL dNTP Mixture (10 mmol / L each), 0.25 μL Taq enzyme (concentration 20 U / μL), and 12.25 μL ddH2O. The final concentration of F and R primers in the reaction system was 5 μM. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; then 36 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds; and finally, extension at 72°C for 10 minutes.

[0098] Judgment rules:

[0099] ① High-fruiting-rate materials: Using the genomic DNA of the sample to be tested as a template, if all 30 InDel-labeled primer pairs from InDel_1 to InDel_30 can amplify DNA fragments, the material to be tested is considered to have a high-fruiting-rate material.

[0100] ② Non-high-fruiting-rate materials: Using the genomic DNA of the sample to be tested as a template, if the primer pair for any of the 30 InDel markers, InDel_1 to InDel_30, fails to amplify a DNA fragment, the material to be tested is considered to be non-high-fruiting-rate material.

[0101] 3. PCR product detection

[0102] The PCR products were detected by agarose gel electrophoresis.

[0103] By interpreting the electrophoresis results, the 30 InDel primer pairs were able to effectively amplify the high-fruiting-rate Leymus chinensis materials, and the ones without electrophoresis bands, that is, the ones that could not be amplified, were low-fruiting-rate materials. Figure 7 The second glue map), T13( Figure 8 The first glue map of T15( Figure 8 The third glue map), T16( Figure 8 4th glue map), T18( Figure 9 The second glue map), T19 ( Figure 9 The third glue map), T21( Figure 10 The first glue map of T28( Figure 12 The first glue map of T29( Figure 12 ) and T30 ( Figure 12 (The third gel image of the PCR amplification assay) There are 10 samples in total, which are marked as having a high fruit setting rate; while the samples that can only be partially amplified are judged to have a low fruit setting rate, that is, samples T1-T9, T11, T12, T14, T17, T20, T22-T27, a total of 20 samples, which are marked as having a low fruit setting rate.

[0104] 4. Verify the fruit set rate of the selected germplasm materials

[0105] Individual spikelets from the 30 selected test materials were collected and air-dried. Ten individual spikelets were then manually sampled to reduce errors. The number of spikelets and seed sets in each spikelet was counted, and the seed set rate was calculated. The results are shown in Table 2. Samples with a seed set rate greater than 65% in the manual sampling were numbered T10, T13, T15, T16, T18, T19, T20, T21, T28, and T29. The seed set rates of all other samples were below 65%, consistent with the MIP marker's results. This double-blind experiment demonstrates the effectiveness of the marker.

[0106] T1-T30 is the F1 generation (father / female) resulting from a cross between Zhongke No. 1 Leymus chinensis (female parent) and Zhongke No. 3 Leymus chinensis (male parent). 100 F1 seeds were randomly selected and planted in PVC cylinders (30 cm in diameter and 50 cm in height). Field sowing was performed to isolate individual plants and cultured continuously for three years. After the fruit set characteristics stabilized in the third year, the fruit set rate of 30 individual plants in the third, fourth, and fifth years was randomly calculated. The statistical method for the fruit set rate of Leymus chinensis in each year was as follows: Five ears were randomly selected from each individual plant to count the number of florets and seeds produced in each ear. The number of seeds produced in each ear was divided by the number of florets per ear to obtain the fruit set rate per ear. The fruit set rate per ear of the five ears was averaged to obtain the fruit set rate for that individual plant for the year. The fruit set rate of Leymus chinensis in the third, fourth, and fifth years was averaged to obtain the fruit set rate of each individual plant.

[0107] Table 2. Statistical results of fruit setting rate

[0108]

[0109]

[0110] In summary, this study, based on transcriptome sequencing data, has developed for the first time a set of MIP molecular markers for distinguishing different seed set rates in Leymus chinensis. The ability to identify Leymus chinensis seedlings with different seed set rates was also validated. The results demonstrated that using 30 primer pairs corresponding to 30 INDEL loci allows for accurate identification of Leymus chinensis resources with different seed set rates, with high efficiency and low cost. This approach can be used for the rapid screening and breeding of high-seed set Leymus chinensis germplasm, making it suitable for widespread application.

[0111] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application of substances for detecting multiple insertion / deletion polymorphisms in the identification or auxiliary identification of the fruit set rate of Leymus chinensis, The substances are respectively named InDel-1-P, InDel-2-P, InDel-3-P, InDel-4-P, InDel-5-P, InDel-6-P, InDel-7-P, InDel-8-P, InDel-9-P, InDel-10-P, InDel-11-P, InDel-12-P, InDel-13-P, InDel-14-P, InDel-15-P, InDel- a composition consisting of these 30 primer pairs: InDel-16-P, InDel-17-P, InDel-18-P, InDel-19-P, InDel-20-P, InDel-21-P, InDel-22-P, InDel-23-P, InDel-24-P, InDel-25-P, InDel-26-P, InDel-27-P, InDel-28-P, InDel-29-P, and InDel-30-P; The InDel-1-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 1 and SEQ ID No. 2; The InDel-2-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 3 and SEQ ID No. 4; The InDel-3-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 5 and SEQ ID No. 6; The InDel-4-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 7 and SEQ ID No. 8; The InDel-5-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 9 and SEQ ID No. 10; The InDel-6-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 11 and SEQ ID No. 12; The InDel-7-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 13 and SEQ ID No. 14; The InDel-8-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 15 and SEQ ID No. 16; The InDel-9-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 17 and SEQ ID No. 18; The InDel-10-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 19 and SEQ ID No. 20; The InDel-11-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 21 and SEQ ID No. 22; The InDel-12-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 23 and SEQ ID No. 24; The InDel-13-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 25 and SEQ ID No. 26; The InDel-14-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 27 and SEQ ID No. 28; The InDel-15-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 29 and SEQ ID No. 30; The InDel-16-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 31 and SEQ ID No. 32; The InDel-17-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 33 and SEQ ID No. 34; The InDel-18-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 35 and SEQ ID No. 36; The InDel-19-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 37 and SEQ ID No. 38; The InDel-20-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 39 and SEQ ID No. 40; The InDel-21-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No.41 and SEQ ID No.42; The InDel-22-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 43 and SEQ ID No. 44; The InDel-23-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 45 and SEQ ID No. 46; The InDel-24-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 47 and SEQ ID No. 48; The InDel-25-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 49 and SEQ ID No. 50; The InDel-26-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 51 and SEQ ID No. 52; The InDel-27-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 53 and SEQ ID No. 54; The InDel-28-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 55 and SEQ ID No. 56; The InDel-29-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 57 and SEQ ID No. 58; The InDel-30-P consists of two single-stranded DNAs with nucleotide sequences of SEQ ID No. 59 and SEQ ID No.

60.

2. A composition for identifying or assisting in identifying the fruit set rate of Leymus chinensis, characterized in that: The composition is the substance described in claim 1.

3. A method for identifying or assisting in identifying the fruit set rate of Leymus chinensis, characterized in that: The method comprises the following steps: using genomic DNA of Leymus chinensis to be identified as a template, performing PCR amplification using each primer pair in the composition described in claim 1, respectively, to obtain PCR products of 30 primer pairs, and identifying the fruit setting rate of Leymus chinensis according to whether the PCR products of the 30 primer pairs have specific bands, wherein the fruit setting rate of Leymus chinensis with specific bands of the PCR products of the 30 primer pairs is higher than that of Leymus chinensis with no specific bands of the PCR products of the 30 primer pairs, and the presence of specific bands in the PCR products of the 30 primer pairs satisfies the following 30 conditions: 1) The PCR product of the InDel-1-P primer pair contains a band of 750-2000 bp; 2) The PCR product of the InDel-2-P primer pair contained a band of 750-2000 bp; 3) The PCR product of the InDel-3-P primer pair contained a band of 750-2000 bp; 4) The PCR product of the InDel-4-P primer pair contains a band of 750-2000 bp; 5) The PCR product of the InDel-5-P primer pair contains a band of 750-2000 bp; 6) The PCR product of the InDel-6-P primer pair contains a band of 750-2000 bp; 7) The PCR product of the InDel-7-P primer pair contains a band of 750-2000 bp; 8) The PCR product of the InDel-8-P primer pair contains a band of 750-2000 bp; 9) The PCR product of the InDel-9-P primer pair contains a band of 750-2000 bp; 10) The PCR product of the InDel-10-P primer pair contains a band of 500-2000 bp; 11) The PCR product of the InDel-11-P primer pair contains a band of 500-2000 bp; 12) The PCR product of the InDel-12-P primer pair contains a 500-2000 bp band; 13) The PCR product of the InDel-13-P primer pair contains a band of 500-2000 bp; 14) The PCR product of the InDel-14-P primer pair contains a band of 500-2000 bp; 15) The PCR product of the InDel-15-P primer pair contains a band of 750-2000 bp; 16) The PCR product of the InDel-16-P primer pair contains a band of 750-2000 bp; 17) The PCR product of the InDel-17-P primer pair contains a band of 750-2000 bp; 18) The PCR product of the InDel-18-P primer pair contains a band of 750-2000 bp; 19) The PCR product of the InDel-19-P primer pair contains a band of 750-2000 bp; 20) The PCR product of the InDel-20-P primer pair contained a band of 750-2000 bp; 21) The PCR product of the InDel-21-P primer pair contained a band of 750-2000 bp; 22) The PCR product of the InDel-22-P primer pair contained a band of 750-2000 bp; 23) The PCR product of the InDel-23-P primer pair contained a band of 750-2000 bp; 24) The PCR product of the InDel-24-P primer pair contained a band of 750-2000 bp; 25) The PCR product of the InDel-25-P primer pair contained a band of 750-2000 bp; 26) The PCR product of the InDel-26-P primer pair contained a band of 750-2000 bp; 27) The PCR product of the InDel-27-P primer pair contained a band of 750-2000 bp; 28) The PCR product of the InDel-28-P primer pair contained a band of 750-2000 bp; 29) The PCR product of the InDel-29-P primer pair contained a band of 750-2000 bp; 30) The PCR product of the InDel-30-P primer pair contained a band of 750-2000 bp; The PCR products of the 30 primer pairs have no specific bands, which means that at least one of the above 30 conditions is not met.

4. The method according to claim 3, characterized in that The annealing condition in the PCR amplification cycle was annealing at 55° C. for 30 seconds.

5. The method according to claim 4, characterized in that The extension condition in the PCR amplification cycle was 72° C. for 60 s.

6. Application, characterized in that, Use of the substance described in claim 1 in identifying or assisting in identifying whether a germplasm material or variety of Leymus chinensis has a high fruit-setting rate during its seedling stage.

7. Application, characterized in that, Use of the substance described in claim 1 in preparing a reagent for identifying or assisting in identifying whether a germplasm material or variety of Leymus chinensis has a high fruit-setting rate during its seedling stage.

8. Application, characterized in that, Use of the substance described in claim 1 in identifying or assisting in identifying whether a germplasm material or variety of Leymus chinensis has a low fruit-setting rate during its seedling stage.

9. Application, characterized in that, Use of the substance described in claim 1 in preparing a reagent for identifying or assisting in identifying whether a germplasm material or variety of Leymus chinensis has a low fruit-setting rate during the seedling stage.

10. A method for breeding Leymus chinensis, characterized in that: The method comprises the steps of using the genomic DNA of the parent Leymus chinensis to be selected as a template, performing PCR amplification using each primer pair in the composition of claim 1, obtaining PCR products of 30 primer pairs, and selecting Leymus chinensis with specific bands in the PCR products of the 30 primer pairs as parents for breeding, wherein the specific bands in the PCR products of the 30 primer pairs meet the following 30 conditions: 1) The PCR product of the InDel-1-P primer pair contains a band of 750-2000 bp; 2) The PCR product of the InDel-2-P primer pair contained a band of 750-2000 bp; 3) The PCR product of the InDel-3-P primer pair contained a band of 750-2000 bp; 4) The PCR product of the InDel-4-P primer pair contains a band of 750-2000 bp; 5) The PCR product of the InDel-5-P primer pair contains a band of 750-2000 bp; 6) The PCR product of the InDel-6-P primer pair contains a band of 750-2000 bp; 7) The PCR product of the InDel-7-P primer pair contains a band of 750-2000 bp; 8) The PCR product of the InDel-8-P primer pair contains a band of 750-2000 bp; 9) The PCR product of the InDel-9-P primer pair contains a band of 750-2000 bp; 10) The PCR product of the InDel-10-P primer pair contains a band of 500-2000 bp; 11) The PCR product of the InDel-11-P primer pair contains a band of 500-2000 bp; 12) The PCR product of the InDel-12-P primer pair contains a 500-2000 bp band; 13) The PCR product of the InDel-13-P primer pair contains a band of 500-2000 bp; 14) The PCR product of the InDel-14-P primer pair contains a band of 500-2000 bp; 15) The PCR product of the InDel-15-P primer pair contains a band of 750-2000 bp; 16) The PCR product of the InDel-16-P primer pair contains a band of 750-2000 bp; 17) The PCR product of the InDel-17-P primer pair contains a band of 750-2000 bp; 18) The PCR product of the InDel-18-P primer pair contains a band of 750-2000 bp; 19) The PCR product of the InDel-19-P primer pair contains a band of 750-2000 bp; 20) The PCR product of the InDel-20-P primer pair contained a band of 750-2000 bp; 21) The PCR product of the InDel-21-P primer pair contained a band of 750-2000 bp; 22) The PCR product of the InDel-22-P primer pair contained a band of 750-2000 bp; 23) The PCR product of the InDel-23-P primer pair contained a band of 750-2000 bp; 24) The PCR product of the InDel-24-P primer pair contained a band of 750-2000 bp; 25) The PCR product of the InDel-25-P primer pair contained a band of 750-2000 bp; 26) The PCR product of the InDel-26-P primer pair contained a band of 750-2000 bp; 27) The PCR product of the InDel-27-P primer pair contained a band of 750-2000 bp; 28) The PCR product of the InDel-28-P primer pair contained a band of 750-2000 bp; 29) The PCR product of the InDel-29-P primer pair contained a band of 750-2000 bp; 30) The PCR product of the InDel-30-P primer pair contains a band of 750-2000 bp.

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