QTL-linked molecular marker for controlling premature senility of corn leaves and application thereof

Abstract

The invention discloses a QTL (quantitative trait loci) linked molecular marker for controlling premature senility of corn leaves. The molecular marker comprises two pairs of primers indel-12 and indel-13, according to the invention, a gene locus related to premature senility is located on a corn chromosome 1 through a BSA-seq technology, and the gene locus is located between two molecular markers indel-12 and indel-13 by developing molecular markers. The two molecular markers can be used for predicting top-to-bottom premature senility of corn leaves.

Description

Technical Field

[0001] The invention belongs to the field of corn genetic breeding and molecular biology, and particularly relates to a QTL-linked molecular marker for controlling corn leaf premature senescence and an application thereof. Background Art

[0002] Leaves are the site of photosynthesis in plants and are also sensitive to plant aging (Zhou Yutang et al., 2016). Aging, as the last stage of plant growth and development, is a process in which plants undergo orderly decomposition and nutrient redistribution (RubertiC et al., 2014). During plant aging, the chlorophyll structure first disintegrates and degrades into various small molecules such as amino acids, sucrose, inorganic phosphorus, etc., which are transported to new tissues and organs, especially seeds, through sieve tubes (Li Zhongpeng et al., 2014). Premature senescence, as an abnormal phenotype of aging, shortens the photosynthesis time and leads to reduced grain yield (Zhang Yanxin et al., 2022).

[0003] Corn is an important crop used as both food and feed and industrial raw material in my country. It is used in food, medicine, and chemical industry. Its planting area ranks first among the three major cereal crops. Its yield affects the country's food security and the development of animal husbandry. Corn yield is closely related to leaf photosynthesis. The late growth period of corn is the critical period for grain formation. Most of the dry matter accumulation in the grain mainly comes from photosynthesis. Leaf senescence has an important impact on corn yield (Li Guanghao et al., 2017). Leaf senescence is a complex and strictly controlled developmental process, which is jointly affected by internal factors and environmental factors. Internal factors include the growth period, regulation of plant hormones and related genes, and environmental factors such as temperature, drought, insufficient light, salt stress, and nutrient deficiency (Qiu Lin et al., 2022). Through the study of senescence-associated genes (SAGs), the potential molecular regulatory mechanisms in the leaf senescence process have been revealed (Kim J et al., 2016). Genes related to premature senescence in corn see2α and see2 β , encoding a cysteine ​​protease, legumin / hemoglobinase, which controls nitrogen distribution during heading and causes leaf senescence (Donnison LS et al., 2007); cloned from maize senescent leaves ccp2 is a cysteine ​​protease gene whose expression increases in naturally senescing leaves and decreases under dark-induced senescence conditions (Griffiths CM et al., 1997); AtNYE1 It is a gene that regulates the activity of chromaffin oxygenase in Arabidopsis thaliana, and plays an important role in regulating chlorophyll degradation during aging, causing yellowing of leaves (Ren G et al., 2007). NEPs) is essential for the biogenesis of photosynthetically active chloroplasts in maize, where NEP The complex consists of RPOTmp Gene encoding, when knocked out RPOTmp Genes that cause delayed chloroplast development in maize during growth and development (Yu QB et al., 2014); maize premature senescence mutant ZmPre2 Based on the results of transcriptional analysis, it is predicted that the loss of glutamine near the C-terminus may cause the plant to age at the beginning of the flowering period (Rajeev G et al., 2020). In addition to corn, early senescence mutants have been found in rice. RLS1 It encodes a NB-containing protein at the carboxyl terminus, and its chlorophyll content rapidly degrades during senescence, leading to premature senescence of leaves (Jiao BB et al., 2012); Rice premature senescence gene LPS1 This leads to an increase in carotenoid content in the body, which in turn increases endogenous ABA, leading to abnormalities in a series of physiological indicators such as H2O2, which may be the cause of its premature aging phenotype (Chu Xiaojie et al., 2021); receptor-like kinases were isolated from aging rice. oeLh , which may cause leaf yellowing by affecting the plant hormone pathway to degrade chlorophyll (Shin NH et al., 2019); ORE1 In Arabidopsis, it interacts with flavonoid transcription factors (GLKs) to promote the synthesis of chlorophyll decomposition genes to promote premature leaf senescence (Rauf M et al., 2013); WRKY71 In Arabidopsis thaliana, it can be directly activated by EIN2 , ORE1 and ACS2 Gene, mediates ethylene signal transduction to promote leaf senescence (YuY et al., 2021); It can be seen that many aging phenomena in plants are genetically controlled and the regulatory network is complex. As an important crop for national food security and animal husbandry development, it is very necessary to develop linked molecular markers that control genes related to premature senescence of corn leaves.

[0004] Molecular markers can quickly and accurately analyze the genetic composition of individuals at the molecular level, thereby predicting phenotypes. In order to eliminate corn germplasm with premature aging as early as possible and create corn germplasm with resistance to premature aging, it is very important to screen out effective detection markers that are closely linked to them based on the study of their genetic mechanisms. Indel markers are especially widely distributed in the genome and are easily detectable and co-dominant, so they are widely used in practice.

[0005] References [1]Donnison LS, Gay AP, Thomas H, et al. Modification of nitrogenremobilization, grain fill and leaf senescencein maize (Zea mays) bytransposon insertional mutagenesis in a protease gene [J]. The Newphytologist, 2007, 173(3): 481-94. [2]Griffiths CM, Hosken SE, Oliver D, et al. Sequencing, expressionpattern and RFLP mapping of asenescence-enhanced cDNA from Zea mays with highhomology to oryzain γ and aleurain[J].Plant Molecular Biology, 1997, 34(5):815-821. [3]Jiao BB, Wang JJ, Zhu XD, et al. A novel protein RLS1 with NB-ARMdomains is involved in chloroplast degradation during leafsenescence in rice[J]. Molecular Plant, 2012, 5(1): 205-222. [4]Kim J, Woo HR, Nam HG. Toward Systems Understanding of LeafSenescence: An Integrated Multi-Omics Perspective on Leaf SenescenceResearch[J]. Molecular plant, 2016, 9(6): 813-25. [5]Rajeev G, Shuping J, Suling Z, et al.The maize prematuresenesence2 encodesfor phytochrome‐dependentlate‐flowering and itsexpressionmodulation improves agronomic traits under abiotic stresses [J].Plant Direct,2020, 4(12): 295-305. [6]Rauf M, Arif M, Dortay H, et al. ORE1 balances leaf senescenceagainst maintenance by antagonizing G2-like-mediated transcription[J]. EmboReports, 2013, 14(4): 382-388. [7]Ren G, An K, Liao Y, et al. Identification of a novel chloroplastprotein AtNYE1 regulating chlorophylldegradation during leaf senescence inArabidopsis[J]. Plant Physiology, 2007,144(3): 1429-1441. [8]Ruberti C, Barizza E, Bodner M, et al. Mitochondria changedynamics and morphology during grapevine leaf senescence [J]. PloS one, 2014,9(7): e102012. [9]Shin NH, Trang DT, Hong WJ, et al. Rice Senescence-InducedReceptor-Like Kinase (OsSRLK) IsInvolved in Phytohormone-Mediated ChlorophyllDegradation[J]. International Journal of Molecular Sciences, 2019, 21(1): 260.

[10] Yu QB, Chao H, Yang ZN. Nuclear-encoded factors associated with the chloroplast transcription machinery of higher plants[J]. Frontiers inPlant Science, 2014, 5(23): 316-326.

[11] Yu Y, Qi Y, Xu J, et al. Arabidopsis WRKY71 regulates ethylene-mediated leaf senescence by directly activating EIN2, ORE1 and ACS2genes [J]. The Plant journal : for cell and molecularbiology, 2021, 107(6): 1819-36.

[12] Qiu Lin, Zhao Linshu, Xie Yongdun, et al. Research progress on plant premature senescence[J]. Journal of Plant Genetic Resources, 2022, 23(02):346-57.

[13] Chu Xiaojie, Lu Tao, Ye Hanfei, et al. Cloning and functional study of rice leaf senescence gene LPS1[J]. Chinese Journal of Rice Science, 2021, 35(05): 427-38.

[14] Li Guanghao, Liu Pingping, Zhao Bin, et al. Effects of controlled-release urea on summer maize yield and leaf senescence characteristics under different water conditions[J]. Chinese Journal of Applied Ecology, 2017, 28(02):571-580.

[15] Li Zhongpeng, Kuai Benke. Research progress on mobilization and recycling of nutrients during green organ aging[J]. Acta Physiologica Sinica, 2014, 50(09): 1322-8.

[16] Zhang Yanxin, Liu Jianmin. Experiment on the yield-increasing effect of delaying the senescence of lower leaves of corn[J]. Agricultural Science and Technology and Information, 2022, (24): 12-4.

[17] Zhou Yutang, Li Yongjun. A review of research on plant leaf senescence[J]. Journal of Hubei University of Engineering, 2016, 36(03): 55-59. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a QTL-linked molecular marker for controlling early senescence of maize leaves and its application.

[0007] The object of the present invention is achieved in the following manner: The molecular markers linked to the QTLs controlling early senescence of maize leaves are characterized in that the molecular markers linked to the QTLs controlling early senescence of maize leaves include two pairs of primers, indel-12 and indel-13, Wherein, the sequence of the primer indel-12 is: F1: 5′-TCATTGGGCGTCTCGTTCAA-3′, SEQ ID No: 1; R1: 5′-AATGCTACAGCCTACAGGGG-3′, SEQ ID No: 2; The sequence of the primer indel-13 is: F2: 5′-ATGCACTGTAGGTAGCTGCC-3′, SEQ ID No: 3; R2: 5′-CCCTCGGTGTTACACCACA-3′, SEQ ID No: 4.

[0008] The molecular markers linked to the QTLs controlling early senescence of maize leaves are used in the identification of early senescence traits of maize leaves.

[0009] The total DNA of the corn to be tested was used as a template, and primers indel-12 and indel-13 were used for PCR amplification. After the amplified products were separated by gel electrophoresis, molecular marker polymorphism data were obtained; if the amplified product had the same target band as the molecular marker indel-12 or indel-13, it could be judged that the probability of the corn leaves to be tested undergoing premature senescence from top to bottom was high; if the amplified product did not have the same target band as the molecular marker indel-12 or indel-13, it could be judged that the probability of the corn leaves to be tested undergoing premature senescence from top to bottom was very low.

[0010] The present invention detects a gene locus related to top-down premature senescence of leaves on 1.0 bin of maize chromosome 1 by the BSA-seq method. By developing indel molecular markers and QTL analysis in the target interval, the gene locus is located between the two molecular markers indel-12 and indel-13. The analysis shows that the polymorphism of the two pairs of molecular markers in maize with different genetic backgrounds can be used to predict the premature senescence trait of leaves.

[0011] Compared with the prior art, the present invention can predict whether corn leaves will age prematurely through the detection of molecular markers, and can eliminate corn germplasm with premature aging at an early stage. It can also be used for molecular marker-assisted breeding of corn with leaves resistant to premature aging, accelerate the breeding process, and create corn germplasm resistant to premature aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 So ldhs1 , L521 and their derived F2 individuals as templates, and PCR amplification was performed using molecular markers indel-12 and indel-13 to obtain the banding pattern of the PCR amplification product; A represents the banding pattern of the individual plant in the F2 population and ldhs1 The banding type is the same, B means the banding type of individual plants in the F2 population is the same as that of the L521 parent, H means the banding type of individual plants in the F2 population is the same as that of the F1 parent, and O means the banding type is missing.

[0013] Figure 2 It is the molecular genetic linkage map of maize and its QTL location map. DETAILED DESCRIPTION

[0014] The inbred line material used in the implementation of the present invention comes from: Progeria inbred lines ldhs1 A maize premature senescence mutant was found in the DH line created by inducing and doubling (L217-1 / L65) using maize haploid breeding technology and named ldhs1 The inbred line L521 is from Zheng 58 / P178, which is an inbred line bred by multiple generations of continuous self-pollination.

[0015] The molecular marker controlling the QTL linkage of early senescence of maize leaves consists of two pairs of primers, indel-12 and indel-13. The sequence of the primer indel-12 is: F1: 5′-TCATTGGGCGTCTCGTTCAA-3′, SEQ ID No: 1; R1: 5′-AATGCTACAGCCTACAGGGG-3′, SEQ ID No: 2; The sequence of the primer indel-13 is: F2: 5′-ATGCACTGTAGGTAGCTGCC-3′, SEQ ID No: 3; R2: 5′-CCCTCGGTGTTACACCACA-3′, SEQ ID No: 4.

[0016] The total DNA of the corn to be tested is used as a template, and PCR amplification is performed with primers indel-12 and indel-13. After the amplified products are separated by gel electrophoresis, molecular marker polymorphism data is obtained; if the amplified product has the same target band as the molecular marker indel-12 or indel-13 marker, it can be judged that the probability of premature senescence of the corn leaves to be tested from top to bottom is high; if the amplified product does not have the same target band as the molecular marker indel-12 or indel-13 marker, it can be judged that the probability of premature senescence of the corn leaves to be tested from top to bottom is very low.

[0017] The QTL linkage molecular marker for controlling corn leaf premature senescence is used for early prediction of corn containing premature senescence genes.

[0018] The embodiments of the present invention will be further described below in conjunction with examples, but the scope of the present invention is not limited thereto and is merely illustrative.

[0019] 1. Preparation of F2 generation plants; Using the superior inbred line L521 with stay-green property as the female parent, the premature senescence mutant ldhs1 The hybrid seed obtained by sowing the F1 generation seeds is the F1 generation plant, the seed obtained by self-pollinating the F1 generation plants is the F2 generation seed. The plant obtained by planting the F2 generation seeds is the F2 generation plant.

[0020] 2. Preliminary location of progeria genes; Thirty individual plants with extreme premature senescence phenotype and 30 individual plants with normal plant phenotype were selected from the F2 population to construct the premature senescence pool and the latest senescence pool, respectively. ldhs1 Parent pool. The HiSeq2500 second-generation sequencing platform was used to carry out on-machine sequencing, with a sequencing depth of 30X. The sequencing results were aligned to the B73 reference genome V5 version to obtain high-quality SNP sites. The genes of premature aging traits were located using the SNP-index algorithm using QTL-seqr (R package), and the premature aging gene was located on chromosome 1, with a physical position range of 23915251~35889095.

[0021] 3. Narrowing of target interval and QTL analysis; To further identify genes related to premature aging, 60 pairs of InDel markers were designed in the target interval using Primer3Plus. The designed markers were cloned in the parent L521 and ldhs1 Perform polymorphic molecular marker screening.

[0022] 10 μL PCR reaction system: 1 μL DNA, 0.75 μL primer, 3.25 μL ddH2O, 5 μL 2×3G TaqMaster Mix for PAGE (Red Dye).

[0023] PCR amplification system: pre-denaturation at 95°C for 5 min; 35 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 40 s; extension at 72°C for 5 min; and storage at 16°C for future use.

[0024] The detection method of the above-mentioned markers is 6% polyacrylamide gel detection. Before electrophoresis, wipe the glass plate with 75% alcohol, and then wipe it with stripping silane and affinity silane respectively. After drying, press and assemble. Add 150μL of 10% ammonium persulfate and 600mL of PAGE polyacrylamide respectively and pour along the glass head. Then insert the comb and set it aside after solidification. The PCR-amplified samples to be tested are respectively placed into the holes of the comb, and electrophoresis is performed for 1 hour at a constant power of 55W. Fixation, silver staining, and development are performed after electrophoresis. The fixation method is: soak in the fixative (distilled water: anhydrous ethanol: glacial acetic acid = 180:20:1) for 5 minutes, wash with distilled water 2-3 times, then soak in the silver stain (1L distilled water + 2g AgNO3) for 11 minutes; wash with distilled water 2-3 times; finally soak in the developer (3L distilled water + 30g NaOH + 20ml formaldehyde). After the DNA band pattern appears, take it out and rinse with clean water, and then read the bands.

[0025] A total of 22 pairs of polymorphic primers were screened, and PCR amplification and electrophoresis were performed on 609 individual plants in the F2 population (the PCR reaction system, PCR amplification system and electrophoresis method were the same as above). After electrophoresis, the polymorphic data of molecular markers were recorded, and the amplified band patterns were as follows: Figure 1 As shown, the band type of individual plants in the F2 population was compared with ldhs1 The same banding pattern was marked as "A", the same banding pattern as the L521 parent was marked as "B", the same banding pattern as the F1 was marked as "H", and the missing banding pattern was marked as "O". Combining the phenotypes of different plants, the software QTL IciMapping4.2 was used to construct a genetic linkage map and detect QTLs. A close linkage with the gene localization gene for leaf premature senescence was detected between the indel-12 and indel-13 markers, such as Figure 2 shown.

[0026] The above is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several changes and improvements can be made without departing from the overall concept of the present invention, which should also be regarded as the scope of protection of the present invention.

Claims

1. Molecular markers linked to QTLs controlling early leaf senescence in maize, characterized in that: The molecular markers controlling the QTL linkage of early senescence of maize leaves include two pairs of primers, indel-12 and indel-13. Wherein, the sequence of the primer indel-12 is: F1: 5′-TCATTGGGCGTCTCGTTCAA-3′, SEQ ID No: 1; R1: 5′-AATGCTACAGCCTACAGGGG-3′, SEQ ID No: 2; The sequence of the primer indel-13 is: F2: 5′-ATGCACTGTAGGTAGCTGCC-3′, SEQ ID No: 3; R2: 5′-CCCTCGGTGTTACACCACA-3′, SEQ ID No:

4.

2. The molecular marker linked to the QTL controlling premature senescence of maize leaves according to claim 1 is used in the identification of premature senescence traits of maize leaves.

3. The method for detecting molecular markers linked to QTLs controlling premature senescence of maize leaves according to claim 2, characterized in that: The total DNA of the corn to be tested was used as a template, and primers indel-12 and indel-13 were used for PCR amplification. After the amplified products were separated by gel electrophoresis, molecular marker polymorphism data were obtained; if the amplified product had the same target band as the molecular marker indel-12 or indel-13, it could be judged that the probability of the corn leaves to be tested undergoing premature senescence from top to bottom was high; if the amplified product did not have the same target band as the molecular marker indel-12 or indel-13, it could be judged that the probability of the corn leaves to be tested undergoing premature senescence from top to bottom was very low.

4. A method for determining molecular markers linked to QTLs controlling premature leaf senescence in maize, characterized in that: A gene locus related to premature leaf senescence from top to bottom was detected on the 1.0 bin of maize chromosome 1. By developing indel molecular markers and QTL analysis in the target interval, the gene locus was located between the two molecular markers indel-12 and indel-13. The analysis showed that the polymorphism of the two pairs of molecular markers in maize with different genetic backgrounds can be used to predict premature leaf senescence traits.

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