Description:FIELD OF INVENTION
The current invention relates to the field of plant breeding. The current invention more specifically relates to novel simple sequence repeat (SSR) markers linked to genetic male sterility (GMS) trait in cotton. These markers can be used to detect, identify and select male sterile cotton plants.
BACKGROUND
Cotton (Gossypium spp.) is the world's most important textile fiber crop and is also one of the world's most important oilseed crops. Cotton plants provide a source of human food, livestock feed, and raw material in industry. Cotton seed is pressed for cooking oil and the residual cottonseed oil meal is used for animal feed. Cotton is not only a world’s leading textile fiber and oilseed crop, but it is also of significance for bioenergy production from cotton plant stalks. Global cotton fiber demand is expected to continue to see an annual growth at a rate of 2-3% through 2030. Such a growth is expected to lead to total fiber demand of close to 142 million tons in 2030. Decreasing arable land, declining water supplies, and the impact of global climate change, makes maintaining increasing production to meet increasing demands uncertain.
Conventional breeding of cotton, which requires large amounts of water and land, cannot sustain such levels of increasing production without other advanced supporting technologies such as new hybrid cotton plants that may have desirable traits such as disease resistant, less water requirement, and higher yield.
Out of the fifty known Gossypium species, four species including G. hirsutum and G. barbadense allotetraploids and two species G. herbaceum and G. arboreum diploids are cultivated. Upland cotton (G.hirsutum), known for long staple cotton, or Mexican cotton, produces over 90 per cent of the world’s cotton (Mehetre S. J. Ref 6).
The genus Gossypium is very large, currently comprised of more than fifty species. Two tetraploid species of Gossypium have spinnable seed fibers called lint. These two species are G. hirsutum (referred to as American Upland cotton) and G. barbadense (referred to as Pima cotton). The upland cotton (Gossypium hirsutum L.) genome (2n=4x=52) is large and complex, requiring a large collection of DNA markers to achieve maximum genome coverage and utility in diverse germplasm. The goal of cotton breeding is to improve cotton plant's performance, and consequently its economic value, by combining various desirable traits into a single plant. Improved performance can include one or more of many desirable traits. For example, higher yields of cotton plants contribute to increased lint fiber production, more profitable agriculture and lower cost of products for the consumer. Improved plant health increases the yield and quality of the plant and reduces the need for application of protective chemicals. Adapting cotton plants to a wider range of production areas achieves improved yield and vegetative growth. Improved plant uniformity enhances the farmer's ability to mechanically harvest cotton.
New cotton varieties can be developed by inbreeding heterozygous plants and selecting for superior plants for several generations until substantially homozygous plants are obtained. During the inbreeding process, the vigor of the plant lines decreases and after a sufficient amount of inbreeding, additional inbreeding merely serves to increase seed of the developed variety. Cotton varieties are typically developed for use in the production of hybrid cotton lines.

A cross between two defined substantially homozygous cotton plant varieties always produces a uniform population of heterozygous hybrid cotton plants. When two different, unrelated cotton parent plant varieties are crossed to produce an F1 hybrid, one parent variety is designated as the male, or pollen parent, and the other parent variety is designated as the female, or seed parent. Because cotton plants are capable of self-pollination, hybrid seed production requires elimination of, or inactivation of pollen produced by the female parent to render the female parent plant male sterile to prevent the cotton plant variety designated as the female from self-pollinating. This process is highly labour intensive and adds highly significant cost to any breeding programs. Different options exist for controlling male fertility in cotton plants such as physical emasculation, genetic male sterility, cytoplasmic male sterility and application of gametocides. Incomplete removal of male parent plants from a hybrid seed production field before harvest provides the potential for unwanted production of self-pollinated or sib pollinated seed, which can be unintentionally harvested and packaged with hybrid seed.
Thus, for self-pollinating crops such as cotton, producing hybrids is a challenging task.
Efficient hybrid seed production requires that cross-pollination predominates over self-pollination. A major limitation in the production of hybrid seed for many crop species including cotton is the lack of simple, reliable and economical methods of generating sterility in at least one parent (especially the male parent, to result in male-sterility while leaving female gametes intact and accessible for pollination by a suitable pollen donor). Male sterility is also useful where pollen spread is not desirable, e.g., from a domestic plant to its wild relatives, or where flower fertilization is not desirable, e.g., in the case of ornamental flowers which deteriorate in condition after pollination.

The development of new cotton plant varieties and hybrid cotton plants is a slow, costly process which requires high amount of breeding expertise. The development of new varieties and hybrid cotton plants involves numerous steps, including: (1) selection of parent cotton plants (germplasm) for initial breeding crosses; (2) inbreeding of the selected plants from the breeding crosses for several generations to produce a series of varieties, which individually breed true and are highly uniform; and (3) crossing a selected variety with an unrelated variety to produce the F1 hybrid progeny having increased vigor.

Molecular markers can be effectively utilized to characterize the vast germplasm resources of Gossypium spp. Molecular breeding integrated with conventional phenotypic selection is increasingly being utilized for key traits in cotton. A critical requirement for success is availability of large number of easily accessible and polymorphic DNA markers for analyzing in cotton breeding populations, so that the breeding process become cost effective. The low level of polymorphism, especially within upland cotton, necessitates that a large library of DNA markers be available for various applications. Identification of quantitative trait loci (QTL) is one such tool to help in marker-assisted selection (MAS) of resistant genotypes.
There are multiple genes governing genetic male sterility in cotton; ms5 and ms6 are two major loci, which together confer genetic male sterility in homozygous double recessive state.
Molecular breeding is nowadays the method of choice for the utilization of molecular (DNA-based) tools, including markers, to enhance the efficiency of the plant breeding process.
Identification of novel molecular markers associated with any desirable trait is a complex process. Identifying molecular markers linked with genetic male sterility in cotton can pave the way for convenient and less time consuming molecular breeding of cotton plants, by making cross-pollination between inbred varieties easier.
Though there are some reports for molecular markers in cotton associated with other traits such as lint quality, photosensitive male sterility, there are not many molecular markers reported to be associated with genetic male sterility in cotton. Some of the reports for cotton molecular markers are US patent no. US9290819B2 (Ref 11) ; and Chinese patent application no. CN103255139B (Ref 12) . Feng et al (Ref 5) described some molecular markers associated with GMS in G.hirsutum.
The current invention discloses novel simple sequence repeats (SSR) markers linked with genetic male sterility in cotton plants, and a method of identifying, selecting and breeding cotton plants exhibiting genetic male sterility associated with ms5ms6 double recessive genes.
SUMMARY
The current invention encompasses a method of identifying and selecting a genetically male sterile cotton plant by detection of SSR markers associated with ms5 and ms6 double recessive genotype in cotton plants.
One embodiment of the current invention is a primer pair for identifying genetic male sterile cotton plant or germplasm by detecting polymorphism at two simple sequence repeat (SSR) markers, one each associated with ms5 and ms6 loci in cotton, wherein the primer pair comprises :
a. Primer pair 1 for detecting polymorphism for an SSR marker SSR1 (TMHC24) associated with ms5 sterile genotype, wherein the primer pair 1 comprises a forward primer given in SEQ ID NO:1 and a reverse primer given in SEQ ID NO: 2;
b. Primer pair 2 for detecting polymorphism for an SSR marker SSR2 (TMHC64) associated with ms6 sterile genotype , wherein the primer pair 2 comprises a forward primer given in SEQ ID NO: 3 and a reverse primer given in SEQ ID NO:4.
In one embodiment, the current invention encompasses the primer pair 1 as disclosed herein, wherein the polymorphism detected for the SSR1 marker comprises presence of either a 210bp sterile marker allele or presence of a 220bp fertile marker allele or both in a cotton plant or germplasm.
In one embodiment, the primer pair 2 as disclosed herein, wherein the polymorphism detected for the SSR2 marker comprises presence of either a 247bp sterile marker allele or presence of a 260bp fertile marker allele or both in a cotton plant or germplasm.
In one embodiment, the SSR1 marker comprises an SSR1 motif with the sequence (AAC)n, wherein n = 3 - 30. In one embodiment, the SSR1 marker allele comprises a SSR1 motif with the sequence (AAC)n, wherein n = 22. In one embodiment, n >30 in some plants.
In one embodiment, the SSR2 marker comprises an SSR2 motif with the sequence (TACA)y- xt - (AC)z; wherein “x” is any nucleotide and wherein t = 1 – 30, y = 3-30, and z = 2-30 . In one embodiment, the SSR2 marker allele comprises an SSR2 motif with the sequence (TACA)y - xt - (AC)z, wherein “x” is any nucleotide, and wherein t = 13, y=14, and z = 13. In one embodiment, y >30 and z > 30 in some plants.

One embodiment of the current invention is a method of identifying a genetically male sterile cotton plant or germplasm using the SSR molecular marker primer pair1 and primer pair 2 as disclosed herein, the method comprising the steps of : detecting in a cotton plant the presence of polymorphism of the SSR molecular markers SSR1 and SSR2 linked with ms5 and ms6 loci respectively, by PCR amplification of the SSR1 and SSR2 motifs in the cotton genome using the primer pair 1 and primer pair 2 respectively, as disclosed herein, wherein the male sterile cotton plant or germplasm is homozygous for both the SSR1 and SSR2 sterile marker alleles .
In one embodiment, the method disclosed herein further comprises the steps of:
a. extracting genomic DNA from cotton plant or germplasm sample;
b. amplifying the SSR1 and SSR2 markers from the genomic DNA isolated in steps (a) by PCR amplification using primer pair 1 and primer pair 2;
c. performing gel electrophoresis on the amplified products; and
d. determining presence of polymorphism of SSR1 and SSR2 markers in the genomic DNA sample based on gel electrophoresis.
In one embodiment, the current invention encompasses the method as disclosed herein, wherein the male sterile homozygous genotype of the SSR1 marker at the ms5 locus is detected by the presence of only 210bp sterile marker allele in the cotton plant or germplasm, as detected by the presence of only 210bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and male sterile homozygous genotype of the SSR2 marker at the ms6 locus is detected by the presence of only 247bp sterile marker allele as detected by the presence of only 247bp amplified product in the amplified genomic DNA of the cotton plant or germplasm sample using the primer pair 2.

In one embodiment, the method disclosed herein further encompasses the method of selecting genetically male sterile cotton plant comprising homozygous sterile marker alleles for both SSR1 and SSR2 markers in the method as disclosed herein.

In one embodiment, the method further comprises the step of identifying a maintainer fertile single heterozygous genotypes of the SSR1 and the SSR2 markers at the ms5 and ms6 loci respectively, in a cotton plant or germplasm, wherein the maintainer fertile genotype is detected by:
a. the presence of both 210bp sterile allele and 220 bp fertile marker allele for SSR1 marker at the ms5 locus as detected by presence of both 210 bp as well as 220bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of single 247bp sterile marker allele for the SSR2 marker at the ms6 locus as detected by the presence of only 247bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2; or
b. the presence of single 210bp sterile allele for the SSR1 marker at the ms5 locus as detected by presence of only 210 bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of both 247bp sterile allele and the 260bp fertile marker allele for the SSR2 marker allele at the ms6 locus as detected by the presence of both the 247bp and the 260bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2.

In one embodiment, the current invention encompasses a method of identifying a male sterile, maintainer fertile, or double heterozygous fertile cotton plant or germplasm from a cotton plant or germplasm population produced by crossing a male sterile cotton plant or germplasm with a fertile cotton plant or germplasm, the method comprising the steps of:
a. Crossing the genetically male sterile plant or germplasm which is homozygous for sterile alleles both the SSR1 and SSR2 markers at the ms5 and ms6 loci respectively, with a second recurrent fertile parent cotton plant or germplasm to obtain a F1 plant and a segregating progeny F2 plant or germplasm population by selfing of the F1 plant;
b. Selecting a F2 progeny plant from the segregating progeny F2 plant population from step (a) for sterile plants, maintainer plants and double heterozygous plants as disclosed herein, wherein the maintainer and double heterozygous plants are fertile;
c. repeating selection step b “n” number of times whenever maintainer plants or double heterozygous plants or germplasm are selected, wherein n is 2 to 8 more filial generations, and wherein the single heterozygous plants and the double heterozygous plants or germplasm are selected, wherein single heterozygous and double heterozygous plants are fertile; and
d. selecting a sib-mating progeny plant or germplasm population derived from sib-crossing a near-isogenic fertile maintainer plant or germplasm that is heterozygous either for SSR1 or SSR2 marker alleles, with a sterile plant or germplasm that is homozygous for both SSR1 and SSR2 sterile marker alleles.

In one embodiment, the method disclosed herein, wherein the second parent plant or germplasm is a recurrent parent containing unfavourable alleles at ms5 and ms6, and the method further comprises the steps :
a. backcrossing the F1 plant or germplasm obtained in step (a) of the method given above, with the recurrent parent cotton plant to get BC1F1 progeny plant population or germplasm;
b. selecting a progeny plant or germplasm from the segregating BC1F1 progeny plant or germplasm population heterozygous for both SSR1 and SSR2 sterile marker alleles at the ms5 and ms6 loci respectively, and backcrossing the selected progeny plant or germplasm with the recurrent parent plant or germplasm to produce BC2F1, wherein the double heterozygous plant or germplasm is fertile;
c. Repeating step (a)- (b) “n” number of times, wherein “n” is 2 to 5 or more, to obtain BCnF1 progeny plant, followed by selfing of BCnF1 plants to get BCnF2 segregating progeny plant or germplasm; and
d. Selecting BCnF2 near-isogenic recurrent plant type progeny plant or germplasm with maintainer plant that is heterozygous for either SSR1 or SSR2 sterile marker alleles at, and sib-mating a near isogenic maintainer fertile plant with a sterile plant within family to produce a male sterile plant or germplasm with background genotype of the recurrent parent plant or germplasm.
BRIEF DESCRIPTION OF FIGURES AND SEQUENCES
Fig. 1 shows a gel image showing ms5 and ms6 markers expected PCR products in maintainer plants of different GMS inbreds which are heterozygous for either ms5 or ms6. ms5 maintainer plants are heterozygous for ms5 loci with presence of 210bp and 220bp for ms5 and 247bp for ms6 loci in homozygous state. ms6 maintainer plants are heterozygous for ms6 loci with presence of 247bp and 260bp for ms6 and 210bp for ms5 loci in homozygous state.
Fig. 2 shows gel image showing ms5 and ms6 marker data for fertile plants 1 to 12 from ms6 maintainer designated GMS inbred R281. All fertile plants are heterozygous at ms6 loci with the presence of 247bp and 260bp for ms6 and homozygous for ms5 loci with 210bp.
Fig. 3 shows gel image showing ms5 and ms6 marker data for sterile plants 13 to 22 from ms6 maintainer designated GMS inbred R281. All sterile plants are double homozygous both at ms5 and ms6 loci with the presence of 210bp and 247bp, respectively.
Fig. 4 shows gel image showing ms5 and ms6 markers segregation in various combinations expected in GMS x fertile population. ms5 marker is expected to show either 210 or 220 or both, and ms6 marker is expected to show either 247 or 260 or both depending individual genetic segregants configuration at ms5 and ms6 loci.
SEQ ID NO:1 is the forward primer for amplifying SSR1 ms5 marker, from primer pair 1.
SEQ ID NO: 2 is the reverse primer for amplifying SSR1 ms5 marker, from primer pair 1,
SEQ ID NO:3 is the forward primer for amplifying SSR2 ms6 marker, from primer pair 2.
SEQ ID NO: 4 is the reverse primer for amplifying SSR2 ms6 marker, from primer pair 2.
DETAILED DESCRIPTION
The current invention discloses novel SSR markers for identifying and selecting cotton plants exhibiting genetic male sterility associated with the presence of ms5ms6 double homozygous recessive genotype. The current invention discloses a gel-based method for identifying and selecting genetically male sterile cotton plants by using specific primers to detect two SSR markers associated with ms5 and ms6 double recessive genotype.
The current invention discloses development and validation of novel gel based markers that are tightly linked to ms5ms6 based genetic male sterility (GMS) trait in cotton which can be used in marker based selection of fertile or sterile phenotypes before flowering and new GMS lines development through trait introgression or breeding
Cotton is a dicot plant with perfect flowers, i.e., cotton has male, pollen producing organs and separate female, pollen receiving organs on the same flower. Because cotton has both male and female organs on the same flower, cotton breeding techniques take advantage of the plant's ability to be bred by both self-pollination and cross-pollination. Self-pollination occurs when pollen from the male organ is transferred to a female organ on the same flower on the same plant. Cross-pollination occurs when pollen from the male organ on the flower of one plant is transferred to a female organ on the flower on a different plant. A plant is sib-pollinated (a type of cross-pollination) when individuals within the same family or line are used for pollination (i.e. pollen from a family member plant is transferred to the stigmas of another family member plant). Self-pollination and sib pollination techniques are traditional forms of inbreeding used to develop new cotton varieties, but other techniques exist to accomplish inbreeding.
For self-pollinating crops such as cotton, producing hybrids is a challenging task.
Efficient hybrid seed production requires that cross-pollination predominates over self-pollination. A major limitation in the production of hybrid seed for many crop species including cotton is the lack of simple, reliable and economical methods of generating sterility in at least one parent (especially the male parent, to result in male-sterility; while leaving female gametes intact and accessible for pollination by a suitable pollen donor). Male sterility is a useful trait also when pollen spread is not desirable, e.g., from a domestic plant to its wild relatives.
Self-pollination for several generations produces homozygosity at almost all gene loci, forming a uniform population of true breeding progeny, known as inbreds. Hybrids are developed by crossing two homozygous inbreds to produce heterozygous gene loci in hybrid plants and seeds.
Male sterility can be accomplished by many methods, such as by physical removal of the organs containing the male gametes. this can be highly labor-intensive and therefore expensive process. Physical emasculation is also difficult in many plant species because of the plant's anatomy. Alternative techniques that do not involve manual or physical emasculation are therefore highly desirable.
Self-incompatibility is a form of infertility caused by the failure of cotton plants with normal pollen and ovules to set seed due to some physiological hindrance that prevents fertilization. Self-incompatibility hinders self-pollination and inbreeding and fosters cross-pollination.
Induction of Male Sterility by Male Gametocides: Male sterility can be induced through the use of chemicals, which are commonly known as male gametocides. Some of the chemicals used for induction of male sterility are FW-450 (Sodium B-Dichloro-iso-butyrate) or MH-30 (Maleic hydrazide) and Ethidium bromide (a potent mutagen).
Chemical gametocides have been also described as a method of generating male-sterile plants. Typically, such a chemical gametocide is a herbicidal compound that when applied to a plant at an appropriate developmental stage or before sexual maturity is capable of killing or effectively terminating the development of a plant's male gametes while leaving the plant's female gametes, or at least a significant proportion of them, capable of undergoing cross-pollination. However, the levels of the chemical necessary to kill most of the male gametes while leaving a sufficient number of female gametes still capable of fertilization can result in undesirable effects such as phytotoxicity.
Spraying of aqueous solution of FW-450 or MH30 induces male sterility in cotton. Application of 2-3 dichloro-iso-butyrate at the rate of 1.02 lb per acre shows selective toxicity to the male gametes. Higher concentration of treatments can cause male as well as female sterility and various adverse effects like reduction in yield, boll and seed size and increase in lint percentage (Singh et al, Ref 9).
Hence, commercial production of hybrid seed using chemical gametocides is limited by their lack of selectivity for gametes, and phytotoxic effects.
Another option for making hybrids is by having inbred parent that comprise a male sterility trait or transgene imparting sterility. This emasculated inbred, often referred to as the female, produces the hybrid seed, F1. The hybrid seed that is produced is heterozygous. However, the grain produced by a plant grown from F1 hybrid seed is referred to as F2 grain. F2 grain which is a plant part produced on the F1 plant will comprise segregating germplasm, even though the hybrid plant is heterozygous. The F1 hybrid plant shows greatly increased vigor and seed yield compared to parent inbreds. Inbred plants on the other hand are mostly homozygous, rendering them less vigorous. Although several genes have been identified, a recessive genic male sterility system based on the ms5 and ms6 alleles seems advantageous to cotton breeders due to its prominent advantages in stable and complete male sterility in hybrid seed production.

Hybrid cotton seed is commonly produced by hand emasculation in one of its parental lines which adds huge labour cost and time. Development of GMS based hybrids in cotton reduces the cost of hybrid seed production by eliminating the hand emasculation process completely. GMS based hybrid seed production can reduce huge labour cost and increase genetic purity. Multiple genes for genetic male sterility trait have been reported in cotton but ms5ms6 double recessive gene based GMS is commonly used for hybrid breeding across world. Conversion of elite fertile lines into GMS recessive homozygous lines through conventional methods needs selfing after each backcross generation to identify double heterozygous fertile plants which leads to higher number of generations in the trait conversion program. Molecular markers linked to GMS could play a critical role to avoid the progeny testing and alternate selfing, and in reducing the breeding cycle in a backcrossing method.
Based on its mode of inheritance, male sterility may be divided into nuclear male sterility (also called genic male sterility; GMS) and cytoplasmic male sterility (CMS). In contrast to CMS and its commercial use for hybrid production, GMS can enhance random mating to develop hybrids since almost all commercial cultivars can be used as its restorer line. The major shortcoming of GMS is that the offspring of GMS plants, pollinated by heterozygous pollinators, theoretically will produce a 1 : 1 male sterile and fertile segregation. Therefore, the fertile offspring have to be scored and eradicated, which is uneconomical if done by traditional means. The use of molecular markers, for marker assisted selection is a very useful tool for identifying and selecting the male sterile progeny.
The pollen sterility that is caused by nuclear genes is termed as genic or genetic male sterility (GMS). In cotton, GMS has been reported in upland, Egyptian and arboreum cottons. In tetraploid cotton, male sterility is governed by both recessive and dominant genes. So far, 19 GMS genes in tetraploid cotton have been identified, including seven single recessive genes (ms1, ms2, ms3, ms13, ms14, ms15 and ms16), four duplicate/ double recessive genes (ms5ms6 and ms8ms 9) and eight single dominant genes (MS4, MS7, MS10, MS11, MS12, MS 17, MS18 and MS19).
However, male sterility governed by recessive genes is used more frequently in plant breeding.
Genetic male sterility is unstable and there are chances of male sterile plants becoming male fertile under low temperature condition. Out of 16 different genes reported in G. hirsutum ms5ms6 is the most stable source. Moreover, in GMS, 50% population is male fertile and the same is identified after flower initiation.
Thus, there is need to use markers in GMS for early identification and removal of fertile plants to save resources and time, quickly convert non GMS lines into GMS versions and use in GMS breeding program to select for desirable combinations to develop new GMS inbred lines.
Male sterility has important applications in the development of hybrids. Molecular marker-assisted selection (MAS) is being applied extensively to cotton.
The identification of GMS linked molecular markers can greatly increase the efficiency of transferring GMS alleles to elite inbred lines within a short period of time using MAS. Genetic mapping of GMS genes is also important for map-based cloning in cotton. Identification of molecular markers closely linked to the ms5 and ms6 alleles is useful for effective transferring of male-sterility genes into cultivars or elite lines using marker-assisted backcrossing, or genetic purity of GMS inbreds. The ms5 and ms6 markers disclosed herein can be used for :
• GMS trait phenotype prediction before flowering or at any stage for hybrid seed production.
• Accelerated GMS line conversion through markers and new GMS lines development through marker assisted breeding
• Accurate quality control or validation of commercial hybrid GMS parental lines for genetic purity.
• Improving the genetic purity of GMS hybrids by GMS markers based QC and elimination of all fertile plants before seed production.
• Elimination of selfing in back cross conventional trait introgression breeding by foreground marker-based advancements.

Maintenance of GMS lines
GMS lines are maintained by sib mating between fertile and sterile plants within family. The pollination is done by hand. The identification of sterile plants can be done by manual selection or by MAS.
Genetic male sterility (GMS) in cotton mediated by the two homozygous recessive genes, ms5ms5 and ms6ms6, is expressed as non-dehiscent anthers and unviable pollen grains. Ms5 ms6 genes are not cloned yet but have been genetically mapped. The approximate physical interval that is known to encompass the ms5 and ms6 genes may vary between different reports. Sequence polymorphism linked to ms5 on A12 and ms6 on D12 can lead to identification of novel markers.
Several molecular markers including SNPs and gel based markers such as SSRs have been reported for ms5ms6 GMS in cotton. SNP markers require high-throughput platform for genotyping and also require stringent haplotype validation in breeding germplasm before deployment. Gel based markers such as SSRs require enough polymorphism resolution to clearly score GMS segregations on the gel. If the resolution is good, gel markers can be deployed directly for breeding populations, trait introgression, forward breeding and GMS female inbred segregation QC studies for commercial hybrid GMS inbred parents.
In the conventional GMS line conversion breeding, double heterozygous plants during backcross F1 (BCnF1) generations can be identified only by testing selfed progeny of all plants. Backcrossing alternating with self-pollination is required to identify plants with the double heterozygous genotype in order to maintain ms5 and ms6 genes in BCnF1 generations. However, markers can completely eliminate this step and identify double heterozygous plants at BCnF1 or any other generations accurately at any stage of the plant
Plants that are heterozygous for either ms5 or ms6 favorable alleles disclosed herein are single heterozygous maintainer plants, and have fertile phenotype. These maintainer plants can be used to maintain the male sterile plants. Plants that are homozygous for both ms5 and ms6 alleles disclosed herein are double homozygous and are phenotypically sterile, and genetically male sterile.
Definitions:
As used herein, the term "cotton plant" or “germplasm” includes whole cotton plants, cotton plant cells, cotton plant protoplast, cotton plant tissue, cotton plant cell or cotton tissue culture from which cotton plants can be regenerated, cotton plant cells that are intact in cotton plants or parts of cotton plants, such as cotton seeds, , cotton flowers, cotton leaves, cotton stems, cotton buds, cotton roots, cotton root tips and the like.
“Self-pollination” occurs when pollen from the male organ is transferred to a female organ on the same flower on the same plant.
“Self-incompatibility” is a form of infertility caused by the failure of cotton plants with normal pollen and ovules to set seed due to some physiological hindrance that prevents fertilization. Self-incompatibility restricts self-pollination and inbreeding and fosters cross-pollination.
“Cross-pollination” occurs when pollen from the male organ on the flower of one plant is transferred to a female organ on the flower on a different plant.
As used herein, a “male sterile plant” is a plant that does not produce male gametes that are viable or otherwise capable of fertilization.
The term “female” refers to a plant that produces ovules. Female plants generally produce seeds after fertilization. A plant designated as a “female plant” may contain both male and female sexual organs or may only contain female sexual organs either naturally or due to emasculation by physical or chemical means, or from male-sterility.
The term “male” refers to a plant that produces pollen grains. The “male plant” generally refers to the sex that produces male gametes for fertilizing ova. A plant designated as a “male plant” may contain both male and female sexual organs, or may only contain male sexual organs either naturally (e.g., in dioecious species) or due to emasculation (e.g., by removing the ovary).
A plant is sib-pollinated (a type of cross-pollination) when individuals within the same family or line are used for pollination (i.e. pollen from a family member plant is transferred to the stigmas of another family member plant).
Self-pollination and sib pollination techniques are traditional forms of inbreeding used to develop new cotton varieties, but other techniques exist to accomplish inbreeding.
GMS lines can be maintained by sibmating between fertile and sterile plants. The pollination is manual. The identification of sterile plants done by trained personnel, and thus 50% plant population is eliminated.
A “maintainer plant” or “maintainer line” as defined herein is a plant that is heterozygous for either ms5 or ms6 and homozygous recessive vice versa for ms5 or ms6.
New cotton varieties are developed by inbreeding heterozygous plants and practicing selection for superior plants for several generations until substantially homozygous plants are obtained. During the inbreeding process with cotton, the vigor of the lines decreases and after a sufficient amount of inbreeding, additional inbreeding merely serves to increase seed of the developed variety. Cotton varieties are typically developed for use in the production of hybrid cotton lines.
Vigor is restored when two different varieties are cross pollinated to produce the first generation (F1) progeny. A cross between two substantially homozygous cotton plant varieties always produces a uniform population of heterozygous hybrid cotton plants and such hybrid cotton plants are capable of being generated indefinitely from the corresponding variety cotton seed supply. When two different, unrelated cotton parent plant varieties are crossed to produce an F1 hybrid, one parent variety is designated as the male, or pollen parent, and the other parent variety is designated as the female, or seed parent. Because cotton plants are capable of self-pollination, hybrid seed production requires elimination of or inactivation of pollen produced by the female parent to render the female parent plant male sterile. This serves to prevent the cotton plant variety designated as the female from self-pollinating.
As used herein, a "polymorphism" is a variation in the DNA between two or more individual plants within a population. A polymorphism preferably has a frequency of at least 1 % in a population. A useful polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an "indel".
As used herein, the terms “marker allele” and “allele” are used interchangeably herein. They refer to alleles for a specific marker. In the current invention the alleles for the SSR1 marker at the ms5 locus and the alleles for the SSR2 marker at the ms6 locus are the “marker alleles”.
SSR (short sequence repeats) or microsatellites are short, tandemly repeated DNA sequence motifs that consist of two to six nucleotide or more core units, and are highly abundant in eukaryotic genome but also occur in prokaryotes at lower frequencies. These small repetitive DNA sequences provide the basis for PCR-based multi allelic, co-dominant genetic marker system. The high incidence of detectable polymorphism through changes in repeat numbers is caused by an intramolecular mutation mechanism called DNA slippage. The regions flanking the microsatellites are generally conserved and PCR primers relative to the flanking regions are used to amplify SSR- containing DNA fragments. The length of the amplified fragment will vary according to the number of repeated residues. Furthermore, the reproducibility of SSRs is very high. In cotton, SSRs have highly accelerated cotton genome mapping work.
An SSR based marker system can be very informative in linkage analysis relative to other marker systems in that multiple alleles may be present. The PCR detection of SSRs is done by use of two oligonucleotide primers flanking the polymorphic segment of repetitive DNA. PCR detection of the SSR markers is usually done by repeated cycles of heat denaturation of the DNA followed by annealing of the primers to their complementary sequences at low temperatures, and extension of the annealed primers with DNA polymerase. The amplified bands are then detected by running them on a gel.
Many SSR marker alleles are linked to a particular desired trait, with a particular number of copies of the repeat occurring in the plants with the desired trait. The plants that do not exhibit the desired trait have a different number of repeats, which can be less than or more than the number of copies found in the plant with the desirable trait. These can be easily identified by PCR amplification and detecting them on a gel.
The heterogeneity of SSR markers makes them advantageous for use as molecular genetic markers. SSR gnomic variability is inherited, is multiallelic, codominant and is reproducibly detectable. Increasing availability of sophisticated amplification-based detection techniques. (e.g. PCR-based) provides several sensitive methods for the detection of nucleotide sequence heterogeneity. Primers (or other types of probes) are designed to hybridize to conserved regions that flank the SSR domain, resulting in the amplification of the variable SSR region. The different sized amplicons generated from an SSR region have characteristic and reproducible sizes. The different sized SSR amplicons observed from two homologous chromosomes in an individual, or from different individuals in the plant population are generally termed “marker alleles”. If there are at least two SSR alleles that produce PCR products with at least two different sizes, the SSR region can be used as a marker.
As used herein, the term “amplification” or “amplifying” used in the context of “nucleic acid amplification” herein is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase-based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods, such as the ligase chain reaction (LCR), and RNA polymerase based amplification (e.g., by transcription) methods. An “amplicon” is an amplified nucleic acid, for example, a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like). The current invention more specifically involves techniques for amplifying DNA molecules such as cotton genome or genomic fragments by any nucleic acid amplification method. The nucleic acid amplification method is PCR in one embodiment of the invention. As used herein, the term "allele" refers to one of two or more different nucleotide sequences that occur at a specific locus.
An allele is "associated with" a trait when presence of the particular allele is part of or linked to a DNA sequence or allele is correlated with the expression of the trait.
An allele "negatively" correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.
An allele "positively" correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
As used herein, a "favourable allele" is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, examples of such traits may be disease resistance, resistance to herbicides, etc; the favourable allele allows the identification of plants with that agronomically desirable phenotype. A favourable allele of a marker is a marker allele that segregates with the favourable phenotype.
As used herein, the term “favorable allele” is used interchangeably with the term “sterile allele”. A favorable or sterile marker allele disclosed herein is associated with either ms5 or ms6 sterile genotype. A double recessive genotype of ms5ms5ms6ms6 is associated with a male sterile phenotype.
As used herein, the double heterozygous plant or germplasm refers to a plant or germplasm having both the ms5 and ms6 sterile marker alleles in heterozygous state. Plants having such genotype are fertile plants.
As used herein, the single heterozygous plant or germplasm refers to a plant or germplasm having one of either the ms5 or ms6 sterile marker alleles in heterozygous state. Plants having such genotype are fertile maintainer plants.
The genotypes of all three genotypes in the cotton plants described herein are given below in Table 1. The small letters denote recessive allele (Ref 3: Chen et al) which as the sterile allele genotype, and the caps letters depict the dominant allele which have fertile allele genotype (for the current invention, the sterile marker alleles are observed by the presence of bands of size approximate 210bp and approximate 247bp respectively; for SSR marker sterile allele at the ms5 and ms6 loci ).
Table 1
Male sterile ms5ms5ms6ms6
Maintainer Ms5ms5ms6ms6 or ms5ms5Ms6ms6,
Double heterozygous Ms5ms5Ms6ms6

As used herein, the term "locus" refers to a position on a chromosome, e.g. where a nucleotide, gene, sequence, or marker is located.
As used herein, the term "marker locus" refers to a specific chromosome location in the genome of a species where a specific marker can be found.
Closely linked loci such as a marker locus and a second locus can display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less.
Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9 %, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.75%, 0.5%, 0.25%, or less) are also said to be "proximal to" each other. In some cases, two different markers can have the same genetic map coordinates. In that case, the two markers are in such close proximity to each other that recombination occurs between them with such low frequency that it is undetectable.
As used herein, the term “molecular markers” or "Genetic markers" refers to nucleic acids that are polymorphic in a population. The term includes nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well- established in the art. These include, e.g., PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).
As used herein, the term, "marker allele", used interchangeably with the term "allele of a marker locus", can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.
As used herein, the term "Marker assisted selection" (of MAS) refers to a process by which individual plants are selected based on marker genotypes. The particular marker genotypes may be linked to specific desirable agronomic traits.
As used herein, the term "Marker assisted counter-selection" refers to a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
As used herein, the terms “foreground selection” and “forward selection” are used interchangeably, and refer to selecting plants having the marker/ favourable allele of the donor parent at the target locus.
As used herein, the term, the term "haplotype" is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype" can refer to alleles at a particular locus, or to alleles at multiple loci along a chromosomal segment.
As used herein, the term "marker haplotype" refers to a combination of marker alleles at a marker locus on same or different chromosome.
As used herein, the term "complement" refers to a nucleotide sequence that is complementary to a given nucleotide sequence.
As used herein, the term "contiguous DNA" refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.
As used herein, the term "heterogeneity" is used to indicate that individuals within the group differ in genotype at one or more specific loci.
As used herein, a centimorgan ("cM") is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
As used herein, the term "chromosomal interval" designates a contiguous linear span of genomic DNA on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly defined or limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. Thus, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
As used herein, the term "closely linked", means that recombination between two linked loci occurs with a frequency of equal to or less than about 10% (i.e., are separated on a genetic map by not more than 10 cM). Thus, the closely linked loci co-segregate at least 90% of the time.
As used herein, the term "probe" refers to a nucleic acid sequence or molecule that can be used to identify the presence of a specific DNA or protein sequence; e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization.
As used herein, the term "Fragment" refers to a portion of a nucleotide sequence.
As used herein, the term "phenotype", "phenotypic trait", or "trait" refer to the observable expression of a gene or series of genes. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., weighing, counting, measuring (length, width, angles, etc.), microscopy, biochemical analysis, or an electromechanical assay.
In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a "single gene trait" or a "simply inherited trait". In the absence of large levels of environmental variation, single gene traits can segregate in a population to give a "qualitative" or "discrete" distribution, which means that the phenotype falls into discrete classes. In other cases, a phenotype is the result of several genes and can be considered a "multigenic trait" or a "complex trait".
As used herein, the term "crossed" or "cross" refers to a sexual cross and involves the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).
As used herein, the term "Backcrossing" refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the "donor" parent refers to the parental plant with the desired gene/genes, locus/loci, or specific phenotype to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed.
As used herein, the term "elite line" refers to any line that has resulted from breeding and selection for superior agronomic performance.
As used herein, the term "genetic map" refers to a representation of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by how frequently their alleles appear together in a population (their recombination frequencies). Alleles can be detected using DNA or protein markers, or observable phenotypes. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. Genetic distances between loci can differ from one genetic map to another. Information can be correlated from one genetic map to another using common markers. One of ordinary skill in the art can use common marker positions to identify positions of markers and other loci of interest on each individual genetic map. The order of loci does change between maps, although frequently there may be small changes in marker orders due to reasons such as markers detecting alternate duplicate loci in different populations, differences in statistical approaches used to order the markers, novel mutation or laboratory error.
As used herein, the term "genetic map location" is a location on a genetic map relative to surrounding genetic markers on the same linkage group where a specified marker can be found within a given species.
As used herein, the term "Germplasm" refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., cotton germplasm collection or Andean germplasm collection). In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
As used herein, the term "haploid" refers to a plant that has a single set (genome) of chromosomes.
As used herein, the term "hybrid" refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
As used herein, the term "inbred" refers to a line that has been bred for genetic homogeneity.
As used herein, the term "indel" refers to an insertion or deletion, wherein one line may be referred to as having an inserted nucleotide or piece of DNA relative to a second line or the second line may be referred to as having a deleted nucleotide or piece of DNA relative to the first line.
As used herein, the term "introgression" refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
The process of "introgressing" is also referred to as "backcrossing" when the process is repeated two or more times.
As used herein, the term "linkage" is used to describe the degree with which one marker locus is associated with another marker locus or some other locus. The linkage relationship between a molecular marker and a locus affecting a phenotype is given as a "probability" or "adjusted probability".
As used herein, the term "linkage disequilibrium" (or LD) refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non- random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51 % to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype.
A marker locus can be "associated with" (linked to) a trait. The degree of linkage of a marker locus and a locus affecting a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype (e.g., an F statistic or LOD score).
As used herein, "linkage equilibrium" describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).
The "logarithm of odds (LOD) value" or "LOD score" (Risch et al, Ref 7) is used in genetic interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage. LOD scores can also be used to show the strength of association between marker loci and quantitative traits in "quantitative trait loci" mapping. In this case, the LOD score's size is dependent on the closeness of the marker locus to the locus affecting the quantitative trait, as well as the size of the quantitative trait effect.
As used herein, the term, "probability value" or "p-value" is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a locus and a phenotype are associated. The probability score can be affected by the proximity of the first locus (usually a marker locus) and the locus affecting the phenotype, plus the magnitude of the phenotypic effect (the change in phenotype caused by an allele substitution). In some aspects, the probability score is considered "significant" or "nonsignificant". In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of association. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1 , less than 0.05, less than 0.01 , or less than 0.001 .
As used herein, the term, "production marker" or "production SSR marker" refers to a marker that has been developed for high-throughput purposes. Production SSR markers are developed to detect specific polymorphisms and are designed for use with a variety of chemistries and platforms.
As used herein, the term, "quantitative trait locus" or "QTL" refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question.
An "allele of a QTL" (or "QTL allele") can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group. An allele of a QTL can be defined by a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. The haplotype is then defined by the unique fingerprint of alleles at each marker within the specified window.
As used herein, the term "reference sequence" or a "consensus sequence" refers to a defined sequence used as a basis for sequence comparison. The reference sequence for the ms5 and ms6 SSR markers disclosed herein refer to sequences obtained by / from Gossypium hirsutum ZJU_v2.1 reference genome (Ref 4 : Hu et al).
As used herein, the terms "agronomic traits", and "plant trait or characteristic" are used interchangeably and refer to the traits and associated genotype that ultimately lead to higher yield but encompass any plant characteristic that can lead to higher plant health and yield, such as herbicide resistance, emergence vigour, vegetative vigour, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, male sterility, threshability and the like.
The current invention encompasses genetic male sterility as the desirable trait for the cotton plants being selected by the method disclosed herein. More specifically, the molecular markers disclosed herein are closely associated with, and help select cotton plants which have the double recessive genotype ms5ms5ms6ms6; and which phenotypically display male sterility.
Marker-assisted selection (MAS):
Molecular markers can be used in a variety of plant breeding applications A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is very useful where the phenotype is hard to assay, for example, disease resistance traits. Since DNA marker assays are less laborious and less time and space- consuming than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line.
The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed.
The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a' perfect marker'.
“Percent (%) sequence identity” with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In certain embodiments, sequence identity may be based on the Clustal V or Clustal W method of alignment.
EMBODIMENTS:
The present invention relates to identifying and selecting cotton plants that are male sterile, and the male sterility is conferred by the presence of ms5ms6 double recessive genes. The current invention encompasses molecular markers for identifying and selecting genetically male sterile cotton plants and all types of genetic configurations expected in any GMS sterile x fertile segregating populations. The invention further relates to methods for generating such cotton plants, as well as the cotton plants generated by the methods disclosed herein. The invention further relates to the use of such male sterile cotton plants in making cotton hybrids by cross-pollinating with other inbred plant varieties. The invention also relates to cotton plants or plant parts thereof, obtained by or obtainable by the method as described herein, as well as cotton plants or plant parts thereof comprising the marker alleles described herein.
The current invention discloses novel gel-based markers tightly linked to genetic male sterility (GMS) trait in cotton which can be used in marker based selection of fertile or sterile phenotypes before flowering or at any stage, and can be used to convert non-GMS lines into GMS lines through marker-assisted selection, and also aid in quality control/ validation of GMS inbred parents of GMS based hybrids during seed production
These markers can be used to convert non-GMS lines into GMS lines, and help in validating the genetic purity of GMS-inbred parents of hybrids.
The current invention encompasses a method of identifying and selecting a genetically male sterile cotton plant by detection of SSR markers associated with ms5 and ms6 double recessive genotype in cotton plants.
One embodiment of the current invention is a primer pair for identifying genetic male sterile cotton plant or germplasm by detecting polymorphism at two simple sequence repeat (SSR) markers, one each associated with ms5 and ms6 loci in cotton, wherein the primer pair comprises :
c. Primer pair 1 for detecting polymorphism for the SSR marker SSR1 (TMHC24) associated with ms5 sterile genotype, wherein the primer pair 1 comprises the forward primer given in SEQ ID NO:1 and reverse primer given in SEQ ID NO: 2;
d. Primer pair 2 for detecting polymorphism for the SSR marker SSR2 (TMHC64) associated with ms6 sterile genotype, wherein the primer pair 2 comprises the forward primer given in SEQ ID NO: 3 and reverse primer given in SEQ ID NO:4.
In one embodiment, the current invention encompasses the primer pair 1 as disclosed herein, wherein the polymorphism detected for the SSR1 marker comprises presence of either a 210 bp sterile marker allele or presence of a 220bp fertile marker allele or both in a cotton plant or germplasm.
In one embodiment, the primer pair 2 as disclosed herein, wherein the polymorphism detected for the SSR2 marker comprises presence of either a 247 bp sterile marker allele or presence of a 260bp fertile marker allele or both in a cotton plant or germplasm.
In one embodiment, the SSR1 marker comprises an SSR1 motif with the sequence (AAC)n, wherein n = 3 - 30. In one embodiment, the SSR1 marker allele comprises a SSR1 motif with the sequence (AAC)n, wherein n = 22. In one embodiment, n >30 in some plants.
In one embodiment, the SSR2 marker comprises an SSR2 motif with the sequence (TACA)y- xt - (AC)z; wherein “x” is any nucleotide and wherein t = 1 – 30, y = 3-30, and z = 2-30 . In one embodiment, the SSR2 marker allele comprises an SSR2 motif with the sequence (TACA)y - xt - (AC)z, wherein “x” is any nucleotide, and wherein t = 13, y=14, and z = 13. In one embodiment, y >30 and z > 30 in some plants.
One embodiment of the current invention is a method of identifying a genetically male sterile cotton plant or germplasm using the SSR molecular marker primer pair1 and primer pair 2 as disclosed herein, the method comprising the steps of : detecting in a cotton plant the presence of polymorphism of the SSR molecular markers SSR1 and SSR2 linked with ms5 and ms6 loci respectively, by PCR amplification of the SSR1 and SSR2 motifs in the cotton genome using the primer pair 1 and primer pair 2 respectively, as disclosed herein, wherein the male sterile cotton plant or germplasm is homozygous for both the SSR1 and SSR2 sterile marker alleles .
In one embodiment, the method disclosed herein further comprises the steps of:
e. extracting genomic DNA from cotton plant or germplasm sample;
f. amplifying the SSR1 and SSR2 markers from the genomic DNA isolated in steps (a) by PCR amplification using primer pair 1 and primer pair 2;
g. performing gel electrophoresis on the amplified products; and
h. determining presence of polymorphism of SSR1 and SSR2 markers in the genomic DNA sample based on gel electrophoresis.
In one embodiment, the gel electrophoresis is done on 3% agarose gel to separate the different DNA bands amplified by PCR of the cotton genomic DNA using the primer pair 1 and primer pair 2.
In one embodiment, the size of the amplified products using primer pair 1 and primer pair 2, from cotton genomic DNA from germplasm or plant sample, can vary by about 3 to 4 bp from the sizes given herein.
It is to be understood by a person of skill in the art that there is some inherent approximation of DNA amplicon sizes when detected by gel electrophoresis.
In one embodiment, the current invention encompasses the method as disclosed herein, wherein the male sterile homozygous genotype of the SSR1 marker at the ms5 locus is detected by the presence of only 210bp sterile marker allele in the cotton plant or germplasm, as detected by the presence of only 210bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and male sterile homozygous genotype of the SSR2 marker at the ms6 locus is detected by the presence of only 247bp sterile marker allele as detected by the presence of only 247bp amplified product in the amplified genomic DNA of the cotton plant or germplasm sample using the primer pair 2.
In one embodiment, the method as disclosed herein, wherein male sterile homozygous genotype for the SSR1 and SSR2 markers at the ms5 and ms6 locus is detected by the presence of allele or PCR product polymorphism on high resolution gel between sizes 200bp to 300bp as visually scored against DNA size marker ladder on gel. The PCR is done with primer pair1 and primer pair 2. In one embodiment, the male sterile homozygous genotype is detected by the presence of a single DNA band (for detecting PCR amplified product) near 210bp by using primer pair 1 for SSR1 marker, and a single band near 247bp by using primer pair 2 for the SSR2 marker.
In one embodiment, fertile plants are detected, by the presence of a single band near 220bp in homozygous state or two bands in heterozygous state, wherein one band is near 210bp and second band near 220bp (210bp/220bp) for the SSR1 marker, and by presence of single band near 260bp in homozygous state or two bands in heterozygous state wherein one band is near 247bp and second band is near 260bp (247bp/260bp) for the SSR2 marker.
In one embodiment, the method disclosed herein further encompasses the method of selecting genetically male sterile cotton plant exhibiting polymorphism for both SSR1 and SSR2 markers in the method as disclosed herein.
In one embodiment, the method further comprises the step of identifying a maintainer fertile single heterozygous genotypes of the SSR1 and the SSR2 markers at the ms5 and ms6 loci respectively, in a cotton plant or germplasm, wherein the maintainer fertile genotype is detected by:
a. the presence of both 210bp sterile allele and 220bp fertile marker allele for SSR1 marker at the ms5 locus as detected by presence of both 210bp as well as 220bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of single 247bp sterile marker allele for the SSR2 marker at the ms6 locus as detected by the presence of only 247bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2; or
b. the presence of single 210bp sterile allele for the SSR1 marker at the ms5 locus as detected by presence of only 210bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of both 247bp sterile allele and the 260bp fertile marker allele for the SSR2 marker allele at the ms6 locus as detected by the presence of both the 247bp and the 260bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2.
In one embodiment, the current invention encompasses a method of identifying a male sterile, maintainer fertile, or double heterozygous fertile cotton plant or germplasm from a cotton plant or germplasm population produced by crossing a male sterile cotton plant or germplasm with a fertile cotton plant or germplasm, the method comprising the steps of:
e. Crossing the genetically male sterile plant or germplasm which is homozygous for sterile alleles both the SSR1 and SSR2 markers at the ms5 and ms6 loci respectively, with a second recurrent fertile parent cotton plant or germplasm to obtain a F1 plant and a segregating progeny F2 plant or germplasm population by selfing of the F1 plant;
f. Selecting a F2 progeny plant from the segregating progeny F2 plant population from step (a) for sterile plants, maintainer plants and double heterozygous plants as disclosed herein, wherein the maintainer and double heterozygous plants are fertile;
g. repeating selection step b “n” number of times whenever maintainer plants or double heterozygous plants or germplasm are selected, wherein n is 2 to 8 more filial generations, and wherein the single heterozygous plants and the double heterozygous plants or germplasm are selected, wherein single heterozygous and double heterozygous plants are fertile; and
h. selecting a sib-mating progeny plant or germplasm population derived from sib-crossing a near-isogenic fertile maintainer plant or germplasm that is heterozygous either for SSR1 or SSR2 marker alleles, with a sterile plant or germplasm that is homozygous for both SSR1 and SSR2 sterile marker alleles.
In one embodiment, the method disclosed herein, wherein the second parent plant or germplasm is a recurrent parent containing unfavourable alleles at ms5 and ms6, and the method further comprises the steps :
e. backcrossing the F1 plant or germplasm obtained in step (a) of the method given above, with the recurrent parent cotton plant to get BC1F1 progeny plant population or germplasm;
f. selecting a progeny plant or germplasm from the segregating BC1F1 progeny plant or germplasm population heterozygous for both SSR1 and SSR2 sterile marker alleles at the ms5 and ms6 loci respectively, and backcrossing the selected progeny plant or germplasm with the recurrent parent plant or germplasm to produce BC2F1, wherein the double heterozygous plant or germplasm is fertile;
g. Repeating step (a)- (b) “n” number of times, wherein “n” is 2 to 5 or more, to obtain BCnF1 progeny plant, followed by selfing of BCnF1 plants to get BCnF2 segregating progeny plant or germplasm; and
h. Selecting BCnF2 near-isogenic recurrent plant type progeny plant or germplasm with maintainer plant that is heterozygous for either SSR1 or SSR2 sterile marker alleles at, and sib-mating a near isogenic maintainer fertile plant with a sterile plant within family to produce a male sterile plant or germplasm with background genotype of the recurrent parent plant or germplasm.
The SSR markers disclosed herein can be used for:
• GMS trait phenotype prediction before flowering or at any stage for hybrid seed production
• Accelerated GMS line conversion through markers and new GMS line development using marker-assisted breeding.
• Accurate quality control or validation of commercial hybrid GMS parental lines for genetic purity
• Improving the genetic purity of GMS hybrids by GMS markers based QC and elimination of all fertile plants before seed production
• Elimination of selfing in back cross conventional trait introgression breeding by foreground marker based advancements.
EXAMPLES
Examples
Example 1: GMS-SSR markers development
Cotton ms5ms6 genes are genetically mapped on chr A12 and chr D12 in public domain but tightly linked markers are not available for large scale breeding deployment with greater accuracies. Moreover, both ms5 and ms6 are not cloned yet to develop functional markers. We located putative candidate regions harbouring ms5 and ms6 loci on chromosome-A12) and chromosome-D12 using bioinformatics approaches by aligning genetic map positions onto to physical map. All possible microsatellite/SSR markers were identified in putative physical regions spanning ms5 and ms6 intervals, and primers were designed using standard approaches for a set of 45 (ms5) and 30 (ms6) high quality SSR markers.
Example 2: GMS SSR marker-trait linkage analysis using BSA
Three GMS sib-mating populations (R52G, R4281G, R42G) were grown in field and phenotypically confirmed for expected fertile and sterile phenotype segregations. All fertile and sterile plants were collected from field and DNA extracted. A set of random 10 plants (DNA) in fertile and sterile categories from each population were separately bulked, and fertile and sterile bulks representing each GMS-population were used to test newly designed SSR (75) markers using modified bulk segregant association study (BSA), Ref 10: Zou, C. et al) representing one random sterile and fertile plant along with fertile and sterile bulks. BSA revealed clear association of two markers which showed expected association pattern in sterile and fertile bulks, and individual controls across populations for ms5 and ms6.
Example 3: Validation of markers identified in BSA using GMS sib-mating donor populations
Two markers identified in BSA were further analysed in all lines of three sib-mating populations (R52G, R4281G, R42G; 402 plants) and association confirmed between expected allele profile and field phenotypes. These two markers (TMHC-24 and TMHC-64) also showed very good association with field phenotypes and co-segregated tightly with ms5 and ms6 loci. Markers also showed precise expected segregation patterns for ms5 or ms6 based GMS-sib mating donor populations, and also clearly discriminated the presence of either ms5 or ms6 maintainer in GMS sib mating donor populations across backgrounds.
Fig. 1 shows a gel image showing ms5 and ms6 markers expected PCR products in maintainer plants of different GMS inbreds which are heterozygous for either ms5 or ms6. ms5 maintainer plants are heterozygous for ms5 loci with presence of 210bp and 220bp for ms5 and 247bp for ms6 loci in homozygous state. ms6 maintainer plants are heterozygous for ms6 loci with presence of 247bp and 260bp for ms6 and 210bp for ms5 loci in homozygous state.
Fig. 2 shows Gel image showing ms5 and ms6 marker data for fertile plants 1 to 12 from ms6 maintainer designated GMS inbred R281. All fertile plants are heterozygous at ms6 loci with the presence of 247bp and 260bp for ms6 and homozygous for ms5 loci with 210bp.
Fig. 3 shows gel image showing ms5 and ms6 marker data for sterile plants 13 to 22 from ms6 maintainer designated GMS inbred R281. All sterile plants are double homozygous both at ms5 and ms6 loci with the presence of 210bp and 247bp, respectively.
Example 4: Validation of GMS-SSR markers in segregating F2 populations
Two markers tightly co-segregating with ms5 and ms6 genes were further validated in 8 segregating F2 populations derived from GMS donors x fertile cross, 17 different GMS donor lines, to identify fertile and sterile plants. Markers were also used to identify the presence of either ms5 or ms6 maintainers or mixture of ms5 and ms6 seeds in 17 GMS lines present in our cotton germplasm, and to assess polymorphic potential of gel based markers for breeding purpose and accuracy identification. Marker classification summary is provided in Table 2
Fig. 4 shows Gel image showing ms5 and ms6 markers segregation in various combinations expected in GMS x fertile population. ms5 marker is expected to show either 210 or 220 or both, and ms6 marker is expected to show either 247 or 260 or both depending individual genetic segregants configuration at ms5 and ms6 loci.
Table 2
A, B are dominant over a and b Size of amplified product Size of amplified product
primer 1 primer2
ms5 ms6
ms5 maintainer-Aabb 210/220 247
ms6 maintainer-aaBb 210 247/260
sterile 210 247
Example 5: Confirmation of marker accuracy for single gene segregating breeding and sib populations
All experiments using single maintainer based GMS-sib populations clearly revealed a tight association of these SSR markers with GMS genes on two different chromosomes. We used two F2 populations derived from fertile inbreds containing only MS5 fertile allele/ gene (sterile ms6 gene fixed during line development program) and GMS-sterile plants from one of the original GMS sib populations. The F2 is expected to segregate only for ms5 locus in 3:1 ratio since ms6 sterile gene is already fixed. GMS flowering data (fertile vs sterile) recorded in these two F2 populations showed 3:1 phenotypic segregation thus confirming the presence of only fertile MS5 gene in one of the inbred line, and ms6 gene in both parental lines. A representative set of 90 confirmed fertile lines from the same population were analysed with both ms5 and ms6 SSR markers. Both populations showed no segregation at ms6 SSR marker locus but clear segregation at ms5 marker locus. Both markers could be able to identify all classes of segregants expected for F2 genetics. Such type of inbred lines where ms6 locus is fixed can be easily used to develop new GMS lines using ms5 markers developed in this study.
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, C , Claims:1. A primer pair for identifying genetic male sterile cotton plant or germplasm by detecting polymorphism for two simple sequence repeat (SSR) markers, one each associated with ms5 and ms6 loci in cotton, wherein the primer pair comprises:
a. Primer pair 1 for detecting polymorphism for an SSR marker SSR1 associated with ms5 sterile genotype, wherein the primer pair 1 comprises a forward primer given in SEQ ID NO:1 and a reverse primer given in SEQ ID NO: 2;
b. Primer pair 2 for detecting polymorphism for an SSR marker SSR2 associated with ms6 sterile genotype , wherein the primer pair 2 comprises a forward primer given in SEQ ID NO: 3 and a reverse primer given in SEQ ID NO:4.
2. The primer pair 1 as claimed in claim 1, wherein the polymorphism for the SSR1 marker comprises presence of either a 210bp sterile marker allele or presence of a 220bp fertile marker allele or both in a cotton plant or germplasm.

3. The primer pair 2 as claimed in claim 1, wherein the polymorphism for the SSR2 marker comprises presence of either a 247bp sterile marker allele or presence of a 260bp fertile marker allele or both in a cotton plant or germplasm.

4. The primer pair as claimed in claim 1, wherein the SSR1 marker comprises an SSR1 motif with the sequence (AAC)n, wherein n = 3-30, and the SSR2 marker comprises an SSR2 motif with the sequence (TACA)y- xt - (AC)z; wherein “x” is any nucleotide and wherein t = 1–30, y = 3-30, and z = 2-30.

5. The primer pair as claimed in claim 4, wherein the SSR1 marker comprises an SSR1 motif with the sequence (AAC)n, wherein n = 22, and the SSR2 marker comprises an SSR2 motif with the sequence (TACA)y – xt - (AC)z; wherein “x” is any nucleotide, and wherein t = 13, y = 14, and z = 13 .

6. A method of identifying a genetically male sterile cotton plant or germplasm using the SSR molecular marker primer pair1 and primer pair 2 as claimed in claim 2, the method comprising the steps of :
detecting in a cotton plant the presence of polymorphism of the SSR molecular markers SSR1 and SSR2 linked with the ms5 and ms6 loci respectively, by PCR amplification of the SSR1 and SSR2 markers in the cotton genome using the primer pair 1 and primer pair 2 respectively, wherein the male sterile cotton plant or germplasm is homozygous for the sterile marker alleles at both the SSR1 and SSR2 markers.
7. The method as claimed in claim 6, wherein the method further comprises the steps of:
a. extracting genomic DNA from cotton plant or germplasm sample;
b. amplifying the SSR1 and SSR2 markers from the genomic DNA isolated in steps (a) by PCR amplification using primer pair 1 and primer pair 2;
c. performing gel electrophoresis on the amplified products; and
d. Determining presence of polymorphism of SSR1 and SSR2 markers in the genomic DNA sample based on gel electrophoresis.

8. The method as claimed in claim 7, wherein male sterile homozygous genotype of the SSR1 marker at the ms5 locus is detected by the presence of only the 210bp sterile marker allele in the cotton plant or germplasm, as detected by the presence of only one 210bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and male sterile homozygous genotype of the SSR2 marker at the ms6 locus is detected by the presence of only the 247bp sterile marker allele as detected by the presence of only a 247bp amplified product in the amplified genomic DNA of the cotton plant or germplasm sample using the primer pair 2.

9. The method of selecting genetically male sterile cotton plant or germplasm comprising homozygous sterile marker alleles for both the SSR1 and SSR2 markers in the method as claimed in claim 6.

10. The method as claimed in claim 7, wherein the method further comprises the step of identifying a maintainer fertile single heterozygous genotypes of the SSR1 and SSR2 markers at the ms5 and ms6 loci in a cotton plant or germplasm, wherein the maintainer fertile genotype is detected by:
a. the presence of both the 210bp sterile marker allele and the 220 bp fertile marker allele for the SSR1 marker at the ms5 locus as detected by presence of both 210bp as well as 220bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of single 247bp sterile marker allele for the SSR2 marker at the ms6 locus as detected by the presence of only 247bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2; or
b. the presence of single 210bp sterile allele for the SSR1 marker at the ms5 locus as detected by presence of a only the 210 bp amplified product in the amplified DNA of the cotton plant or germplasm sample using the primer pair 1, and the presence of both 247bp sterile marker allele and the 260bp fertile marker allele for the SSR2 marker at the ms6 locus as detected by the presence of both 247bp and the 260bp amplified products in the amplified DNA of the cotton plant or germplasm sample using the primer pair 2.

11. A method of identifying a male sterile, maintainer fertile, or double heterozygous fertile cotton plant or germplasm from a cotton plant or germplasm population produced by crossing a male sterile cotton plant or germplasm with a fertile cotton plant or germplasm, the method comprising the steps of:
a. Crossing the genetically male sterile plant or germplasm which has homozygous sterile marker alleles for both the SSR1 and SSR2 markers, with a second recurrent fertile parent cotton plant or germplasm to obtain a F1 plant and a segregating progeny F2 plant or germplasm population by selfing of the F1 plant;
b. Selecting a F2 progeny plant from the segregating progeny F2 plant population from step (a) for sterile plants, maintainer plants and double heterozygous plants as claimed in 9, wherein the maintainer and double heterozygous plants are fertile;
c. repeating selection step b “n” number of times whenever maintainer plants or double heterozygous plants or germplasm are selected, wherein n is 2 to 8 more filial generations, and wherein the single heterozygous plants and the double heterozygous plants or germplasm are selected, wherein single heterozygous and double heterozygous plants are fertile; and
d. selecting a sib-mating progeny plant or germplasm population derived from sib-crossing a near-isogenic fertile maintainer plant or germplasm which has heterozygous sterile and fertile marker alleles either for SSR1 or for SSR2 marker, with a sterile plant that has homozygous sterile marker alleles for both the SSR1 and SSR2 markers.

12. The method as claimed in claim 11, wherein the second parent plant or germplasm is a recurrent parent comprising fertile marker alleles for both the SSR1 and SSR2 markers at the ms5 and ms6 loci respectively, and the method further comprises the steps:
a. backcrossing the F1 plant or germplasm obtained in step (a) of claim 10, with the recurrent parent cotton plant to get BC1F1 progeny plant population or germplasm;
b. selecting a progeny plant or germplasm from the segregating BC1F1 progeny plant or germplasm population heterozygous for both SSR1 and SSR2 markers, and backcrossing the selected progeny plant or germplasm with the recurrent parent plant or germplasm to produce BC2F1, wherein the double heterozygous plant or germplasm that is heterozygous for both SSR1 and SSR2 markers is fertile;
c. Repeating step (a)- (b) “n” number of times, wherein “n” is 2 to 5 or more, to obtain BCnF1 progeny plant, followed by selfing of BCnF1 plants to get BCnF2 segregating progeny plant or germplasm; and

d. Selecting BCnF2 near-isogenic recurrent plant type progeny plant or germplasm with maintainer plant that is heterozygous for either SSR1 or SSR2 markers, and sib-mating a near isogenic maintainer fertile plant with a sterile plant within family to produce a male sterile plant or germplasm with background genotype of the recurrent parent plant or germplasm.