阅读说明：本技术 水稻多雌蕊基因LOC_Os03g11630的分子标记方法 (Molecular marking method of rice multi-pistil gene LOC _ Os03g11630 ) 是由 梁永书 闫超 段文静 南文斌 秦小健 张汉马 于 2020-06-05 设计创作，主要内容包括：水稻多雌蕊基因LOC_Os03g11630的分子标记方法,以多雌蕊水稻为基因供体构建F<Sub>2</Sub>遗传定位分离群体,以F<Sub>2</Sub>群体隐性单株为基因定位群体,借助SSR和InDel<Sup>#</Sup>标记进行PCR扩增,根据PCR扩增电泳结果,对水稻多雌蕊进行高密度连锁分析,获得了与之紧密连锁的基于PCR反应的2对特异PCR引物对L3-135和RM7576,2特异标记间物理距离为40.617Kb,与LOC_Os03g11630基因紧密连锁。当标记L3-135和RM7576能单独或者同时扩增250bp和206bp大小的片段,则该水稻材料携带多雌蕊基因LOC_Os03g11630,并由此获得该多雌蕊基因。本发明用于检测、筛选水稻多雌蕊基因,为水稻遗传机理解析提供基因资源,挖掘与新创优异水稻资源,支持超高产水稻新品种的培育。(Molecular marking method of rice multi-pistil gene LOC _ Os03g11630, and F is constructed by using multi-pistil rice as gene donor 2 Genetically mapping the segregating population with F 2 Recessive individual plant of the population is a gene-located population by virtue of SSR and InDel # The markers are subjected to PCR amplification, and high-density linkage analysis is carried out on the rice pistils according to the PCR amplification electrophoresis result, so that 2 pairs of specific PCR primer pairs L3-135 and RM7576 which are closely linked with the markers and are based on PCR reaction are obtained, the physical distance between the 2 specific markers is 40.617Kb, and the markers are closely linked with the LOC _ Os03g11630 gene. When the markers L3-135 and RM7576 can amplify fragments of 250bp and 206bp separately or simultaneously, the rice material carries multiple pistil genes LOC _ Os03g11630, and the multiple pistil genes LOC _ Os03g11630 are obtained from the rice materialObtaining the multiple pistil gene. The invention is used for detecting and screening the rice multi-pistil gene, provides gene resources for analyzing the rice genetic mechanism, excavates and creats new excellent different rice resources, and supports the cultivation of new varieties of ultrahigh-yield rice.)

1. A molecular marking method of a rice multi-pistil gene LOC _ Os03g11630 is characterized in that: construction of F Using Multi-pistil Rice as Gene Donor₂Genetically mapping the segregating population with F₂Recessive individual plant of the population is a gene-located population by virtue of SSR and InDel^#The markers are subjected to PCR amplification, high-density linkage analysis is carried out on the rice pistils according to the PCR amplification electrophoresis result, 2 pairs of specific PCR primer pairs L3-135(SEQ ID NO: 1, 2) and RM7576(SEQ ID NO: 3, 4) which are closely linked with the markers and are based on PCR reaction are obtained, the physical distance between the 2 specific markers is 40.617Kb, and the 2 specific markers are closely linked with the LOC _ Os03g11630 gene.

2. The method of claim 1 for obtaining a novel rice mullsistylester gene LOC _ Os03g11630, wherein F is constructed by combining rice mullsistylester 172 with rice 02428₂Genetically segregating the population as the subject by F₂The band type data of the L3-135(SEQ ID NO: 1, 2) and RM7576(SEQ ID NO: 3, 4) molecular markers of the population recessive single plant population are used for PCR amplification of the genomic DNA of the rice material to be detected, and when the markers L3-135 and RM7576 can singly or simultaneously amplify fragments with the sizes of 250bp and 206bp, the rice material carries the multi-pistil gene LOC _ Os03g11630, and the multi-pistil gene is obtained.

Technical Field

The invention relates to a molecular marking method for controlling a rice multi-pistil gene LOC _ Os03g11630, belonging to the field of rice excellent gene resource mining and molecular genetics.

Background

Rice is the most important first food crop in the world, more than half of the global population takes rice as staple food, and the rice plays an important role in guaranteeing sustainable food supply in China. Reviewing the research and development process of rice genetic breeding since the country is built in China, the successful cultivation of each high-quality and high-yield new variety and the related paper of rice published in each top-grade journal in the world can not avoid the excavation of excellent germplasm resources of rice and the innovative utilization of specific genes. Therefore, continuous excavation and innovative utilization of excellent rice resources are indispensable important links for breeding new varieties of ultra-high yield rice, and are more source power for enriching and developing basic theoretical researches of rice genetics. The floral organs are indispensable organs in the whole life history of rice, and whether the floral organs are normally developed directly determines the yield and quality of rice. The identification of the related genes affecting the organ number and the structural variation of the rice flowers lays a foundation for further researching the molecular mechanism of the rice flower development and improving the yield and the quality of the rice and excavating the potential germplasm resources of the rice.

Molecular genetics research shows that the rice floral organ variation gene belongs to the quality character controlled by a single gene. With the wide application of gene location cloning technology, the map location cloning technology is one of the most effective means for location cloning of important agronomic trait QTL/gene from rice germplasm resources. By utilizing the marker technologies such as SSR, InDel, Caps and the like and rice floral organ mutation resources from different variation sources, a large number of rice floral organ mutation genes are identified by comprehensively applying a multi-disciplinary and crossed basic theory technology system such as classical genetics, molecular biology, genetic engineering, breeding science and the like, so that the understanding of people on the rice floral organ genetic mutation mechanism is promoted to a certain extent. But do notBecause the previous research selects different types of floral organ mutant materials as gene donors, the genetic difference of the screened and identified target genes is large, the genetic diversity of the genes is rich, and particularly, the physical position distribution of the currently identified floral organ number and structural mutant genes on chromosomes has large difference. So far, related genes for controlling the number of rice floral organs and morphological structure variation are reported, and partial floral organ mutant genes are finely positioned and cloned and are positioned on different chromosomes of rice. At present, a plurality of rice pistil-related genes are not discovered. The applicant draws on 'Hot Jing 35/Xiqingzao B' F₂Screening 1 part of floral organ mutation material from the genetic segregation population, after two continuous multi-generation selfing genetic segregation and purification in Hainan and Chongqing, the floral organ mutants have stable field phenotype and are not influenced by environment, the mutation material shows that 6 stamens are normal in the flowering period, anther filaments are normal in development, pistil mutation shows 2 or 3 ovaries, fertilization and fructification in the maturation period form complete 2 or 3 grains, mature seeds germinate into conjoined double seedlings, and the temporary name of the mature seeds is multi-pistil 172. The applicant uses multiple pistils 172 as gene donors to construct a '02428/multiple pistils 172' hybrid combination to obtain F₀Hybrid seed, planting F₀Obtaining F from hybrid seed₁Plant, F₁Plant selfing to obtain F₂And (2) genetically positioning group seeds, positioning the multiple pistil genes in an L3-135-RM7576 interval on the short arm of the 3 rd chromosome by adopting a map-based positioning cloning strategy, wherein the physical distance between 2 pairs of specific PCR markers is 40.617Kb, and the 2 pairs of specific PCR markers and the multiple pistil genes LOC _ Os03g11630 have a close linkage relation.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a new gene which influences the change of the number of pistils of rice. And provides a molecular marking method and a detection method of the multiple pistil mutant gene.

The technical scheme of the molecular marking method of the rice multi-pistil gene LOC _ Os03g11630 comprises the following steps: the method comprises the steps of constructing an F2 genetic localization segregation population by taking multi-pistil rice as a gene donor, performing PCR amplification by taking a recessive single plant of an F2 population as a gene localization population by means of SSR and InDel # markers, performing high-density linkage analysis on the multi-pistil rice according to the result of PCR amplification electrophoresis, and obtaining 2 pairs of specific PCR primer pairs L3-135(SEQ ID NOS: 1 and 2) and RM7576(SEQ ID NOS: 3 and 4) which are closely linked with the multi-pistil rice based on PCR reaction, wherein the physical distance between the 2 specific markers is 40.617Kb, and the 2 pairs of specific PCR primer pairs are closely linked with LOC _ Os03g11630 genes.

The invention uses 2 pairs of specific PCR markers L3-135(SEQ ID NO: 1, 2) and RM7576(SEQ ID NO: 3, 4) to PCR amplify the genomic DNA of the rice to be detected, if the markers L3-135 and RM7576 can singly or simultaneously amplify fragments with the sizes of 250bp and 206bp, the rice material has a multi-pistil gene LOC _ Os03g 11630.

The molecular marking method of the rice multi-pistil gene LOC _ Os03g11630 or the method for judging whether the rice contains the rice multi-pistil gene LOC _ Os03g11630 is applied to rice floral organ mutation selection.

The rice multiple pistil gene LOC _ Os03g11630 is located on 1 gene site related to rice ovary number mutation on the short arm of the 3 rd chromosome of rice, and the gene can control the number of rice pistils.

The invention utilizes the rice multi-pistil mutant material created by the applicant as a gene donor, and the mutant material is derived from a 'hot japonica 35/synqingzao B' hybrid combination F2 genetic segregation population. In the heading and flowering stage of rice, 1 part of multi-pistil mutant material is obtained by a large number of field phenotype identifications and named as multi-pistil 172, and the multi-pistil 172 is used as a gene donor material.

Compared with the prior rice floral organ mutant gene, the invention has the following characteristics:

1. the gene LOC _ Os03g11630 is obtained by screening multiple pistil mutants by utilizing indica-japonica hybrid progeny groups as a gene donor material, and a new gene is detected by analyzing through a gene map cloning technology. The gene is related to the formation of rice multiple pistils.

2. The gene LOC _ Os03g11630 is positioned on the short arm of the 3 rd chromosome, has no chromosome collinearity overlap with the reported rice floral organ mutant gene, and is a brand new gene. The novel gene can provide gene resources for analyzing the genetic mechanism of the rice floral organs.

Drawings

FIG. 1 is a field phenotype of multiple pistils 172; a. the flowering stage shows more pistils; b-c, forming 2 or 3 complete grains in the mature period; d. and germinating the mature seeds to form conjoined double seedlings.

FIG. 2 shows the multiple pistil genes LOC _ Os03g11630 close linkage markers L3-135, lane 1 is Marker banding pattern; lane 2 is the paternal multiple pistil 172 banding pattern; lane 3 is maternal 02428; lanes 1, 2, 3 … … …, 20, 21, and 22 show the F2 recessive population single-plant electrophoretic band pattern constructed from 02428/multiple pistils 172.

FIG. 3 shows the close linkage marker RM7576 for the multiple pistil gene LOC _ Os03g11630, lane 1 showing the band type of male parent multiple pistils 172; lane 2 is female parent 02428, and lane 3 is Marker banding pattern; lanes 1, 2, 3 … … …, 20, 21, and 22 are the single-plant electrophoretic band pattern of the recessive population "02428/multiple pistils 172" F2.

Detailed Description

The invention is further illustrated below with reference to specific examples of implementation. The method used therein is a conventional method unless otherwise specified.

Control of preliminary mapping of novel genes of rice multiple pistils

1. Test material

The multiple pistils 172 are derived from a 'hot japonica 35/synqingzao B' F2 genetic segregation population, the field performance is shown in figure 1, the mutant material shows that 6 stamens are normal in the heading and flowering stage of rice, anther filaments are normal in development, pistil mutations show 2 or 3 ovary (figure 1-a), the mature seeds are fertilized and fructified to form complete 2 or 3 rice grains (figures 1-B and c) in the wax ripening and mature stage, and the mature seeds germinate into conjoined double seedlings (figure 1-d) and are temporarily named as the multiple pistils 172.

The F2 genetic population constructed by '02428/multiple pistils 172' is taken as the main research material. The construction process is as follows: the F0 seeds are obtained by the hybridization combination of '02428/multiple pistils 172', the pistils of F1 plants develop normally, and the multiple pistil characters show recessive genetic expression. F0 seeds are planted in Hainan Lingshui county, F1 plants are selfed to obtain F2 group seeds, 5000F 2 single plants are planted in Chongqing season, the fact that single pistil (normal) and multiple pistil (mutation) of the F2 single plants meet the Mendelian genetic separation ratio of 3:1 is investigated, and multiple pistil genes are controlled by 1 pair of recessive single genes, so that the method is suitable for developing research on gene map location cloning strategies. 02428 is prepared from Eriocheir sinensis grain of Yunnan japonica rice and Gebang grain of Shanghai japonica rice by hybridizing, and has wide affinity to indica rice and japonica rice.

DNA extraction, PCR amplification and 8% gel electrophoresis

2.1DNA extraction Using SDS method, whole genome DNA was extracted from the recessive F2 individual. The method comprises the following specific steps:

1) leaves of about 4cm are taken and put into a mortar, and ground into powder in liquid nitrogen to a centrifugal tube of 1.5 ml.

2) Adding preheated 65 deg.C extract 600-800ul, mixing, and keeping in 65 deg.C water bath for 20-30 min while turning upside down for 3 times.

3) The centrifuge tube was removed from the water bath and mixed with 200ul of 5mM KAC solution in ice bath for 20-30 minutes.

4) Add 24: 1 chloroform/isoamyl alcohol 600ul, upside down for several times until the lower liquid phase is dark green and centrifuged at 10000rpm for 10 minutes.

5) Taking the supernatant, putting the supernatant into another 1.5ml centrifuge tube, adding frozen ethanol with the volume of 1.5 times of that of the centrifuge tube, slowly and uniformly mixing, standing for 15 minutes at room temperature, and centrifuging for 5 minutes at 10000 rpm.

6) Sucking supernatant, washing with 70% ethanol for 2 times, dehydrating with anhydrous ethanol, and air drying.

7) Adding 400ul ddH₂And storing in a refrigerator at-20 ℃ for later use.

2.2 PCR amplification

According to the reference map, 159 pairs of SSR primers which are uniformly distributed in the whole genome and have polymorphism between parents are selected. Genomic DNA of 22 recessive F2 individuals was randomly selected for Polymerase Chain Reaction (PCR).

1) The total volume of the PCR reaction system was 20ul, and the components are shown in Table 1

TABLE 120 ul PCR reaction System Components

Reagents and templates	Amount of the composition used
		Template DNA	2μl
10×PCR buffer with Mg2+	2μl
		Primer F(10μM)	1μl
Primer R(10μM)	1μl
		dNTP	1μl
Taq enzyme	0.1μl
		ddH2O	12.9ul
Total	20ul

2) The PCR reaction program is:

1) pre-denaturation at 94 ℃ for 3min

2) Denaturation at 94 ℃ for 30s

3) Annealing at 56 deg.C for 30s

4) Extension at 72 ℃ for 1min

30 cycles from step 2 to step 4, and a final extension of 10min at 72 ℃. PCR reactions were run on a Bio-rad Burley model T100 gradient PCR instrument, USA.

2.38% polyacrylamide gel electrophoresis detection

The formula of the 8% polyacrylamide gel working solution is shown in table 2, and the PCR amplification product is electrophoresed by 8% polyacrylamide gel, and the specific steps are as follows:

1) and pouring the prepared gel between two glass plates, immediately inserting a comb into the gel chamber (to prevent gel solidification) after the gel is filled, placing the glass plates in a flat groove, and standing at room temperature for 30min or more until the gel is completely polymerized.

2) After the gel was completely solidified, 1 × TBE buffer was added to the electrophoresis tank, and the comb was carefully pulled out in parallel.

3) Add 3. mu.l bromophenol blue indicator into 10. mu.l PCR reaction product, mix them evenly, take 3ul sample per tube with micro liquid inlet device.

4)200V constant voltage electrophoresis for 1.5 h.

TABLE 28% Polyacrylamide gel working solution

Composition (I)	Amount of the composition used
		10×TBE(ml)	10
40% acrylamide (ml)	20
		Sterilized water (ml)	69
Ammonium persulfate (μ l)	900
		TEMED(μl)	100
Total(ml)	100

3 preliminary positioning

A small F2 recessive single plant population constructed by 02428/multiple pistils 172 is subjected to linkage analysis by using a polymorphic marker, and if 14 single plant banding patterns of 22 recessive single plants are consistent with the banding patterns of a recessive parent by referring to a parental PCR amplification band, the marker is proved to have linkage relation with the recessive parent. The marker RM3467 on the short arm of the 3 rd chromosome amplifies 22 recessive individuals, 17 individuals have the same banding pattern with the multiple pistil 172, the rice multiple pistil gene and the marker RM3467 on the 3 rd chromosome have a linkage relationship, and the multiple pistil gene is preliminarily positioned on the short arm of the 3 rd chromosome.

(II) control of Fine localization of novel Gene of Rice Polypistil

1. Test material

The multiple pistils 172 are derived from a 'hot japonica 35/synqingzao B' F2 genetic segregation population (figure 1), the mutant material shows normal expression of stamens in the heading and flowering stage of rice, 6 stamens and anther filaments develop normally, pistil mutation shows 2 or 3 ovary (figure 1-a), the mature seeds are fertilized and fructified to form complete 2 or 3 rice grains (figures 1-B and c) in the wax ripening and mature stage, and the mature seeds germinate into conjoined double seedlings (figure 1-d) and are named as the multiple pistils 172 temporarily.

2. Fine positioning method

2.1 primer development

According to the sequence difference of sequenced japonica rice varieties Nipponbare, indica rice variety 9311 and Xieqingzao B in the interval compared by SSR primers published on the Gramene website and bioinformatics, further designing SSR and InDel # primers to amplify parents, and screening to obtain polymorphic molecular markers for fine positioning.

2.2 Fine localization method and obtaining of closely-linked molecular markers

Similarly, the F2 genetic population constructed by '02428/mullein 172' is utilized to further expand the number of recessive individuals of the positioning population to 364, primers developed by a target chromosome segment are utilized to screen the exchange individuals of the target chromosome segment, thus constructing a high-density genetic physical map of the target segment, finally, the mullein gene is finely positioned in the L3-135-RM7576 marking segment (figures 2 and 3), 2 pairs of specific PCR primers L3-135(SEQ ID NO: 1 and 2) and SEQ ID NO: 3 and 4) are utilized, the L3-135 and RM7576 are respectively utilized to amplify segments with the sizes of 250bp and 206bp, the L3-135 is marked with reference to the physical position 6048599bp of the Nipponbare genome, the L7576 is marked with reference to the physical position 6079216bp of the Nipponbare marked, the physical distance of the 2 pairs of specific PCR marking segments is 40.617Kb, the L3-135(6048599bp) is only 1 heterozygous individual (individual number 39) of the RM, the marker RM7576(6079216bp) had 1 heterozygous individual (individual No. 137 #). It is fully demonstrated that the rice multi-pistil gene LOC _ Os03g11630 is finely positioned between markers RM7576 and L3-135, thereby obtaining molecular markers closely linked to LOC _ Os03g11630 and amplifying only the fragment carrying the genotype from multi-pistil 172.

(III) identification of multiple pistil candidate genes

The physical position information is marked by markers L3-135 and RM7576 for fine positioning of the rice multi-pistil new gene, only one candidate gene LOC _ Os03g11630 is screened from a (http:// ensemble. gramene.org/Oryza _ sativa/Location/View.

The information of the rice multi-pistil gene LOC _ Os03g11630 fine positioning marker exchange individual plant genotype related chromosome, marker, genotype, physical position and the like is shown in Table 3. There were 13 heterozygous individuals for marker RM5444(5591402bp), 5 heterozygous individuals for marker L3-118(6012517bp), and only 1 heterozygous individual for marker L3-135(6048599bp) (accession # 39). There were 14 heterozygous individuals for marker L3-52(6812449bp), 3 heterozygous individuals for marker L3-97(66239662bp), and 1 heterozygous individual for marker RM7576(6079216bp) (accession No. 137 #). Fully indicates that the rice multi-pistil gene LOC _ Os03g11630 is finely positioned between markers RM7576 and L3-135, and the physical distance between 2 specific markers is 40.617 kb.

TABLE 3 Rice multiple pistil Gene LOC _ Os03g11630 Fine localization marker exchange Individual genotype

Chromosome	3	3	3	3	3	3	3	3	3	3
											Marking	RM5444	RM3467	L3-118	L3-132	L3-135	RM7576	L3-95	L3-97	L3-101	L3-52
Physical location (bp)	5591402	6003496	6012517	6043090	6048599	6079216	6127475	6239662	6240244	6812449
											11	1	1	1	1	1	1	H	H	H	H
14	H	1	1	1	1	1	1	1	1	1
											18	H	H	H	1	1	1	1	1	1	1
20	1	1	1	1	1	1	1	1	1	H
											30	1	1	1	1	1	1	1	1	1	H
39	H	H	H	H	H	1	1	1	1	1
											46	1	1	1	1	1	1	1	1	1	H
71	1	1	1	1	1	1	1	1	1	H
											105	H	1	1	1	1	1	1	1	1	1
137	1	1	1	1	1	H	H	H	H	H
											139	H	1	1	1	1	1	1	H	H	H
145	1	1	1	1	1	1	1	1	1	H
											151	H	H	H	1	1	1	1	1	1	1
157	1	1	1	1	1	1	1	1	1	H
											176	H	1	1	1	1	1	1	1	1	1
207	1	1	1	1	1	1	1	1	1	H
											231	H	H	H	1	1	1	1	1	1	1
235	1	1	1	1	1	1	1	1	1	H
											245	1	1	1	1	1	1	1	1	1	H
275	H	H	H	1	1	1	1	1	1	1
											296	1	1	1	1	1	1	1	1	1	H
329	H	1	1	1	1	1	1	1	1	H
											348	1	1	1	1	1	1	1	1	1	1
349	1	1	1	1	1	1	1	1	1	1
											356	H	1	1	1	1	1	1	1	1	1
359	H	1	1	1	1	1	1	1	1	1
											362	H	1	1	1	1	1	1	1	1	1

Note: 1: multiple pistil 172 genotype; h: a heterozygous genotype; physical position (bp): reference to a clear genomic sequence.

The information of the closely linked markers LOC _ Os03g11630, which are genes of the rice multiple pistils of the present invention, is shown in Table 4. The marker L3-135 information is 6048599bp in the Nipponbare genome position; SEQ ID NO: the length of the sequence 1 and the sequence 2 are both 22bp, the PCR amplification product is 250bp, the annealing temperature is 59.3 and 59.0 ℃, and the primers are Nipponbare and 93-11 insertion deletion (InDel #). The marker RM7576 information is 6079216bp at the position of the Nipponbare genome; SEQ ID NO: the 3 and 4 sequences are 21bp in length, the PCR amplification product is 206bp, the annealing temperatures are 59.1 and 60.2 ℃ respectively, the SSR primers are used, and the repetitive motif is (GCA) 10.

TABLE 4 Rice Multi-pistil Gene LOC _ Os03g11630 close linkage marker information

Physical position (bp): reference is made to the japanese nitrile genome sequence.

Sequence listing

<110> university of Chongqing teacher

<120> molecular marking method of rice multi-pistil gene LOC _ Os03g11630

<130>1

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

cggagcttct gttcatgtgt c 21

<210>2

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gactgcttct tgtacgacga c 21

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ctgccctgcc ttttgtacac 20

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gcgagcattc tttcttccac 20