CN101260430B

CN101260430B - Method for appraising rice fragrance

Info

Publication number: CN101260430B
Application number: CN2008100152649A
Authority: CN
Inventors: 姚方印; 李广贤; 姜明松; 李润芳; 张晓东; 王文英
Original assignee: SHANDONG RICE RESEARCH INSTITUTE
Current assignee: SHANDONG RICE RESEARCH INSTITUTE
Priority date: 2008-04-18
Filing date: 2008-04-18
Publication date: 2011-01-12
Anticipated expiration: 2028-04-18
Also published as: CN101260430A

Abstract

The invention relates to a method for identifying aroma in rice and a molecular marker co-segregated from the aroma characters, belonging to the molecular genetics field. Taking 1 to 2 young leaves from the upper part of a single-stand rice during the tillering stage, storing the single-stand rice in an icebox at -80 DEG C for further use; taking 2-4cm rice leaves and filling into a 1.5ml centrifugal tube, filling in liquid nitrogen, mulling, filling in 900ul TPS solution, water bathing for 20 minutes at 75 DEG C; opening the cover and smelling by nose can precisely judge the existence of aroma. The molecular marker is obtained by PCR amplifying the specificity PCR primer LO1 or LO2. The invention is specially used for the breeding of aromatic rice and genetics of rice aroma, and cloning aroma gene.

Description

A kind of method of identifying rice scent

Technical field

The present invention relates to a kind of method of rice scent of identifying and reach and the common isolating molecule marker of fragrance proterties, belong to the molecular genetics field, be exclusively used in the seed selection and the genetic research of rice scent kind, and can be used for cloning scent gene.

Background technology

Fragrance is the important food flavor quality trait of paddy rice.Many good rice varieties have pleasant fragrance, enjoy human consumer's welcome, and each Rice Production state or area all have fragrant seed rice to plant in the world.

Present measuring method to fragrance mainly contains chews seed method, potassium hydroxide method, high performance liquid chromatography, preceding two kinds of methods are undertaken by sense of smell, chew when the seed method is identified in enormous quantities and can impact tongue and nose, potassium hydroxide method also has certain limitation to the release of fragrance, identifies inaccurate.High performance liquid chromatography provides objective method for the mensuration of fragrance, but this method needs large quantities of sample sizes, and time-consuming (Jin et al., 2003).

Aspect fragrant rice seed selection, utilize molecular marker assisted selection can be accurately, simply, the fragrant rice of seed selection apace.Many investigators study the genetic analysis and the location of scent gene, and for fragrant rice breeding provides some useful molecule markers, but the present molecule marker distance of utilizing is far away, can not accurately identify fragrance.

Up to the present, major part studies show that rice aroma is subjected to a recessive single-gene control.Ahnet al. (1992) utilizes a pair of near isogenic line Lemont and fragrant Lemont, and the scent gene of fragrant Lemont is from donor Della, with savory isolating F ₃Colony is a material, shows by rflp analysis, and scent gene is chain with the RG28 that is positioned on the 8th karyomit(e), and genetic distance is 4.5cM.Li Xin etc. (1995) are material with indica type and round-grained rice phenotypic marker gene, with the fragrant Xian in Wujin, the fragrant round-grained rice hybridization in Wujin, measure parents and F with alkaline leaching respectively ₁, F ₂, and part F ₃The leaf perfume (or spice) of combination, the result shows that scent gene is chain with the marker gene v-8 performance that is positioned on the 8th karyomit(e), estimates that both recombination values are about 38.03% ± 3.84%.Jin etal. (1995) F of CT9993/KDML105 ₂Segregating population is a material, finds a RAPD primer, this primer 1.5kb fragment that only in non-fragrant colony, increases, and performance and fragrance proterties close linkage, with this primer to two parents, F ₂Segregating population individual plant and other different varieties are analyzed, and have further proved this polymorphic reliability.Lorieux et al. (1996) is a material with DH colony, utilize the concentration of RFLP, RAPD, STS and isoenzyme mark and gas Chromatographic Determination AcPy, confirmed that AcPy is the main component of fragrance, and scent gene and RG28 are linked, distance is 5.8cM, identify 2 QTL simultaneously, one is positioned at the 4th karyomit(e), and another is positioned at the 12nd karyomit(e).Garland et al. (2000) has developed a microsatellite marker SCU-RICE-SSR-1 according to the polymorphic regions of RG28 between a pair of fragrant rice and non-fragrant rice varieties, and select 4 SSR marks of near zone 24 fragrant rice and non-fragrant kind to be carried out the polymorphic quantity of information analysis of mark, studies show that SCU-RICE-SSR-1, RM42 and RM223 can be used for distinguishing the multiple allelomorphos of the scent gene in the different varieties.Jin et al. (2003) has found a SNP mark RSP04 chain with scent gene, with the F of RSP04 to 50 individual plants formations ₂Segregating population carries out gene type assay, and finding has two recombinant strains between RSP04 and the scent gene, and RSP04 and scent gene are at a distance of 2cM; With the F of RSP04 to 163 individual plants formations of another one ₂Segregating population carries out gene type assay, and finding has 6 recombinant strains between RSP04 and the scent gene, and RSP04 and scent gene are at a distance of about 2cM.Dong etal. (2001) shows that to the trisome genetic analysis of three local fragrant rice varieties of Japan the fragrance of these three fragrant rice varieties is controlled by a recessive gene, all belongs to allelotrope, is positioned on the 8th karyomit(e).Siddiqet al. (1986) carries out the trisome genetic analysis to India's kind, and finding has two recessive scent genes, lays respectively at the 5th and the 9th karyomit(e).Chen etc. (2006) are positioned at the scent gene seat in the interval of a 69kb.Shi Junqiong (2004) utilizes the single slice substitution line that scent gene has been carried out Primary Location, in two segregating populations scent gene all with RM223 be divided into from, be in the segregating population of donor with Basmati 385, scent gene is respectively 2.7cM and 3.3cM apart from both sides mark RM515 and RM284, be in the segregating population of donor with U.S.'s jasmine perfume (or spice), scent gene is respectively 2.9cM and 11.8cM apart from both sides mark RM515 and RM210.

Utilize molecule marker that the technology of the molecular marker assisted selection of rice scent gene is all had report both at home and abroad, but up to now, be total to isolating mark about rice scent gene and yet there are no report, utilize TPS solution to identify that the fragrance proterties also is to report for the first time.

The fragrant rice aboundresources of China, from different rice varieties, excavate, identify and the location scent gene, find the fragrance proterties method accurately and reliably of identifying, overcome present chew-in technique, KOH method and instrumental method are identified shortcoming inaccurate in the fragrance, set up isolating scent gene molecule marker altogether, and utilization and scent gene altogether isolating molecule marker carry out assistant breeding, judge that scent gene is to exist with isozygotying with heterozygosis, can be chosen in the excellent proterties individual plant that has that isozygotys on the scent gene seat with heterozygosis exactly, and can targetedly scent gene be selected in the laboratory to obtain, improve the efficient of selecting greatly.

Summary of the invention

The objective of the invention is to overcome the existing weak point of identifying the method for rice scent, and a kind of method of identifying rice scent is provided, and provide a kind of and fragrance proterties isolating molecule marker altogether, improve seed selection efficient for molecule marker carries out assistant breeding.

This kind of the present invention identifies that the method for rice scent is as follows:

(1) the rice individual plant top 1-2 sheet spire of fetching water in tillering phase is stored in-80 ℃ of refrigerators standby;

(2) get the long rice leaf of 2-4cm and put into the 1.5ml centrifuge tube, put into liquid nitrogen, grind, add TPS solution 900 μ l, 75 ℃ of water-baths 20 minutes; Wherein TPS solution is: 100mM Tris-HCl (pH8.0), 10mM EDTA (pH8.0), 1M KCl;

(3) open lid, hear with nose, promptly decidable fragrance has or not.

Provided by the present invention and fragrance proterties be isolating molecule marker altogether, be by

Specific PCR primer L01

Upstream extremity is classified CCTTTGTGGTATTCCGTTGTT as from 5 '-3 ' nucleotides sequence

Downstream end is classified GTGTGCACTGGTCACTGTTTT as from 5 '-3 ' nucleotides sequence

Or specific PCR primer L02

Upstream extremity is classified ATGTTTTAGTGTTTTTTCAGGCT as from 5 '-3 ' nucleotides sequence

Downstream end is classified CCAGACATAAATAATGGCATCTT as from 5 '-3 ' nucleotides sequence

Obtain through the pcr amplification oryza sativa genomic dna.Wherein primer L01 amplifies the specific fragment of 280bp in fragrant round-grained rice 9407, amplifies the specific fragment of 271bp in IRBB60.Primer L02 amplifies the specific fragment of 210bp in fragrant round-grained rice 9407, amplify the specific fragment of 200bp in IRBB60.

Utilize the method for evaluation rice scent provided by the invention that the fragrant rice in Qufu, the fragrant rice of open fire, fragrant round-grained rice 9407 (are public kind, country's seed bank all has preservation, can externally provide) respectively with rice varieties IRBB60 (public kind, the country seed bank preservation is all arranged, can externally provide) hybridization F ₂Segregating population carries out the fragrance macroscopical identification.The rice individual plant top 1-2 sheet spire of fetching water in tillering phase is stored in-80 ℃ of refrigerators standby.Get the long rice leaf of 2-4cm and put into the 1.5ml centrifuge tube, put into liquid nitrogen, grind, add TPS solution 900 μ l, 75 ℃ of water-baths 20 minutes.Open lid, hear, can accurately judge having or not of fragrance with nose.TPS solution formula: 100mM Tris-HCl (pH8.0), 10mM EDTA (pH8.0), 1M KCl.

The inventor has designed 2 pairs of specific PCR primers in addition, and it can carry out mark to rice seedling fragrance proterties and identify whether detection has scent gene fgr, and the accuracy height of detection is simple to operate.Because the fragrant rice in Qufu, the fragrant rice of open fire, fragrant round-grained rice 9407 are parents of the Hybrid of widespread usage, therefore this molecule marker also has the universal significance whether detection has the fragrance proterties, can be used as other with the testing tool of fragrant rice varieties as parent's breeding material.

This kind of the present invention identified the method for rice scent and is total to isolating molecule marker with the fragrance proterties, have the following advantages:

(1) utilize the present invention to identify that the method (TPS method) of rice scent identifies rice scent, simple, accurately convenient.Utilize the KOH method can not accurately judge the individual plant of fragrance proterties, utilize the TPS method accurately to judge.

(2) mark is stable, and is not affected by environment.By the specific PCR primer of the present invention's design, detected with this two mark in the different growing sampling in 2007, the mark performance is stable, not affected by environment.

(3) the assistant breeding select target is clear and definite, saves cost.By the amplification of PCR mark, can judge that scent gene is with the individual plant that isozygotys or the heterozygous state genotype exists, accelerate fragrance trait molecular marker assistant breeding, improve the efficient of selecting greatly.

Description of drawings

Fig. 1: the F of 01 pair of fragrant round-grained rice 9407 * IRBB60 hybridization of label L ₂For the molecular marker assisted selection electrophoretogram

M is Marker, P1, fragrant round-grained rice 9407; P2, IRBB60; 1-20, F ₂Separate individual plant.

Fig. 2: the F of 02 pair of fragrant round-grained rice 9407 * IRBB60 hybridization of label L ₂For the molecular marker assisted selection electrophoretogram

Embodiment

1, F ₂The establishment of colony and phenotypic evaluation

Utilized in 2005 the fragrant rice in Qufu, the fragrant rice of open fire, fragrant round-grained rice 9407 respectively with IRBB60 hybridization, the end of the year F ₁Seed send the Sanya, Hainan Island to add generation, and selfing produces F ₂Seed.2006 with F ₂Segregating population is seeded in Shandong Rice Research Institute experimental farm, sowing on May 1, rice transplanting on June 20, individual plant transplanting.20 days extracting F of rice transplanting ₂The individual plant DNA of segregating population, and utilize the present invention to identify the fragrance proterties of method (TPS method) the evaluation paddy rice individual plant of rice scent, divide savory and two kinds of phenotypes of British plain spirits, each segregating population is planted 3000 strains, totally 9000 strains.

2, F ₂The molecular marker analysis of segregating population

(1) F of hybridizing with the fragrant rice/IRBB60 in CTAB method extraction Qufu, open fire fragrant rice/IRBB60, fragrant round-grained rice 9407/IRBB60 ₂Each individual plant DNA.

(2) ssr analysis is with reference to the method for Panaud et al. (1996).20 μ l PCR reaction systems are:

Template DNA 10ng/ μ l, 0.15 μ l 10mM dNTPs, the Taq enzyme of 1.5 units, 2 μ l, 10 * PCRbuffer (contains MgCl ₂), 2uM forward and the reverse primer of 1.5 μ l, reaction volume 20 μ l.The PCR response procedures is: 94 ℃ of pre-sex change 5min of DNA, and 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, and last 72 ℃ are extended 5min.Increase on the PTC-200PCR instrument, amplified production carries out separation electrophoresis on 6% polyacrylamide gel, and silver dyes the observation that develops the color of method with reference to (2002) such as Li Wentao, record experimental result.

(3) rice scent gene fgr has been positioned in (Chen et al.2006, Fen, 2005) between paddy rice the 8th karyomit(e) RM1109 and the PSM465.On this basis, utilize RM1109 and PSM465 to three combination F ₂9000 individual plants of segregating population increase, and screen 106 exchange strains between two marks altogether, utilize the present invention to identify the fragrance of method (TPS method) the evaluation paddy rice exchange strain of rice scent.While is according to the genome sequence of region Japan warm and fine 9311, (upstream extremity is classified CCTTTGTGGTATTCCGTTGTT as from 5 '-3 ' nucleotides sequence to L01 to utilize Primer Premier Version5 design PCR primer, downstream end is classified GTGTGCACTGGTCACTGTTTT as from 5 '-3 ' nucleotides sequence) and L02 (upstream extremity is classified ATGTTTTAGTGTTTTTTCAGGCT as from 5 '-3 ' nucleotides sequence, downstream end is classified CCAGACATAAATAATGGCATCTT as from 5 '-3 ' nucleotides sequence), increase.The result shows L01 and L02 performance polymorphism between the fragrant rice in Qufu, the fragrant rice of open fire, fragrant round-grained rice 9407 and IRBB60, wherein at the amplification of fragrant round-grained rice 9407 and IRBB60 such as accompanying drawing 1,2 shown in.By analyzing genotype and the fragrance phenotype of exchange strain, do not find that exchanging appears in this two mark and scent gene fgr, show be divided into from.Therefore L01 and L02 two marks can be used for screening fragrant rice varieties.

Claims

1. method of identifying rice scent is characterized in that step is as follows:

(2) get the long rice leaf of 2-4cm and put into the 1.5ml centrifuge tube, put into liquid nitrogen, grind, add TPS solution 900 μ l, 75 ℃ of water-baths 20 minutes; Wherein TPS solution is: 100mM Tris-HCl, 10mMEDTA, 1M KCl; The PH of described Tris-HCl is 8.0, and the PH of EDTA is 8.0;

(3) open lid, hear with nose, promptly decidable fragrance has or not.